Cd127 expression inversely correlates with foxp3 and suppressive function of cd4+ tregs

ABSTRACT

The invention provides methods of isolating CD 127lo/− immunosuppressive regulatory T cells which can be greatly enriched for FoxP3, methods of expanding the isolated cells, pharmaceutical compositions of such cells, and methods of their use in the treatment of autoimmune and other immune system mediated disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/958,638, filed Apr. 20, 2018, which is a divisional of U.S.application Ser. No. 14/666,772, filed Mar. 24, 2015, now U.S. Pat. No.9,977,021, issued May 22, 2018, which is a continuation of U.S.application Ser. No. 11/756,479, filed May 31, 2007, now U.S. Pat. No.9,012,134, issued Apr. 21, 2015, which claims the benefit of U.S.Provisional Application Ser. No. 60/803,623 filed May 31, 2006, each ofwhich are incorporated by reference in their entireties for allpurposes.

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The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Feb. 24, 2023, isnamed 081906-1326635-171840US_SL.xml and is 47,808 bytes in size.

BACKGROUND OF THE INVENTION

Over the past decade, there have been tremendous advances in ourunderstanding of the basic process that control immune tolerance. Theidentification of regulatory T cells (Treg), particularly CD4+CD25+Tregs, as an important component of self-tolerance has opened a majorarea of investigation in immunology, and numerous studies havedemonstrated the potent influence of Tregs in suppressing pathologicimmune responses in autoimmune diseases, transplantation, andgraft-vs-host disease (reviewed in (1-6)). Tregs have a unique androbust therapeutic profile. The cells require specific T cell receptor(TCR)-mediated activation to develop regulatory activity but theireffector function appears to be non-specific, regulating localinflammatory responses through a combination of cell-cell contact andsuppressive cytokine production (7-9). Moreover, there are a number oftherapeutic interventions that appear to promote Treg development andfunction (10, 11). This so called “adaptive” regulatory T cellpopulation shares many of the attributes of thymic-dependent, naturalTregs but can differ in critical cell surface biomarkers and functionalattributes (12). For instance, Tr1 and Th3 cells have been describedthat produce IL-10 and TGF, respectively (13, 14). These results haveled to novel approaches to immunotherapy as the ability to isolate andexpand this cell subset in mice has led to novel therapeuticinterventions in immunological diseases (6, 15). However, a majorobstacle to the study and application of Tregs in the human setting hasbeen the lack of specific cell surface biomarkers to define and separateTregs from other regulatory or effector T cell subsets.

Although many studies indicate that CD25 is a crucial cell-surfacemarker for the regulatory subset (16, 17), unlike the mouse, severalstudies have suggested that only the CD4+ T cell subset expressing thehighest levels of CD25 (termed CD25hi) have in vitro suppressiveactivity (16). Moreover, the addition of other markers such as HLA-DRsuggest even a lower percentage (often less than 1%) of CD4+ T cellscomprise the suppressive T cell subset. Finally, some markers such asCTLA-4 and GITR, which have been reported to be expressed on Tregs(18-21), are also expressed on potent effector T cells and as such makeimmunophenotyping and determination of their functional role problematic(22, 23). This has led to a number of disparate reports of Tregquantification in disease settings. For instance, some studies suggestthat the number of CD4+CD25hi Tregs are deficient in Type 1 Diabetes(T1D) (24) while others suggest that the number and function of thesecells is normal in T1D (25). Moreover, the ability to isolate onlylimited numbers of these cells from peripheral blood has made expandingthis regulatory cell population problematic.

One significant advance in the study of mouse and human Tregs has beenthe discovery of the transcription factor, FoxP3, as a major marker andfunctional regulator of Treg development and function (26-29). In aseries of elegant mouse and human genetic studies, investigatorsdemonstrated that mutations in the FoxP3 gene were linked to theautoimmune manifestations observed in the Scurfy mouse and humans withimmune dysregulation, polyendocrinopathy, enteropathy, X linked syndrome(IPEX) disease (28). Subsequent studies in the mouse showed that FoxP3deficient animals lack Tregs while over-expression of the FoxP3 proteinleads to profound immune suppression (30). Although recent studies havequestioned whether all Tregs are FoxP3+ or whether all FoxP3+ T cellsare regulatory, FoxP3 protein remains the best and most specific markerof Tregs to date (30).

In this regard, flow cytometric and immunohistochemical analyses thatFoxP3 is expressed in significantly more T cells than previouslyidentified using the other available cell surface markers, includingCD25. FoxP3 protein is found in CD25 low and negative CD4+ T cells and,under certain conditions, some CD8+ T cells (30, 31). Thus, it is likelythat many of the natural and adaptive regulatory T cells are missed incurrent biomarker studies, calling into question the conclusions relatedto deficiencies or defects in certain autoimmune settings. Importantly,as FoxP3 is an intracellular protein, it cannot be used to separatehuman Tregs for functional studies or for in vivo expansion for cellulartherapy, limiting its use in the human setting.

As noted above, the emergence of Tregs as an essential pathway inmaintaining immune tolerance has opened the opportunity for a betterunderstanding of immune homeostasis and the potential for therapeuticintervention. However, the human phenotyping of Tregs has been complex.Typically, investigators have noted that the most suppressive Tregscoincide with the CD4+ T cells with the brightest CD25 staining.Recently, Cozzo et al (see, J Immunol. 2003 Dec. 1; 171(11):5678-82)have reported that CD4+CD25+ regulatory T cells express low levels ofCD127 in a transgenic mouse. Harnaha et al. have reported in the contextof Type 1 diabetes data indicating that CD4+CD25+ T cells express higherlevels of CD127 (IL-7R alpha) than CD4+CD2S− cells. However, theseresults were again derived from mice, specifically NOD-SCID micereconstituted with ex vivo engineered dendritic cells and NODsplenocytes (see, Harnaha, J et al. Diabetes 2006 January;55(1):158-70). Unfortunately, the ability to accurately gate for CD25 israther arbitrary as no other cell surface marker can be used todefinitively identify the subset. Recently, Baecher-Allen has suggestedthat other markers such as HLA-DR allows for subdividing the CD4+CD25hisubset to enrich Treg activity even further. However, this additionalmarker suggests that the number of Tregs is even less than previouslysuggested (41).

The identification of FoxP3 as a specific transcription factor thatmarks these suppressive T cells suggests that there may be a largerpopulation of Tregs in human peripheral blood than previouslyappreciated, although this has been controversial due to unanticipatedexpression of FoxP3 in a number of activated CD25− T cell populations(30, 38). In fact, there may be regulatory cells that are Foxp3 negativeas well. However, these studies have been compromised by the absence ofcell surface markers that can be used to isolate these and other T cellsubsets to examine regulatory T cell activity since FoxP3 cannot be usedas a means to purify the cells for function.

This invention provides for these and other needs by using the reducedexpression of the CD127 T-cell surface marker as a useful surrogate foridentifying regulatory T-cells which are highly likely to beimmunosuppressive FoxP3+ regulatory T-cells. Use of the CD127 biomarkeralone or in conjunction with other biomarkers can account for up to 7-8%of CD4+ T cells, providing yields significantly greater than identifiedby previous approaches. Moreover, CDI2?1^(◯1)− cells suppress theproliferative response of alloreactive T cells in an MLR and arethemselves anergic to the same stimuli. This is true in spite of thefact that in most individuals only 20-40% of the CD4+CD127lo/− cells areFoxp3+″

BRIEF SUMMARY OF THE INVENTION

The invention relates to the discovery that CD127 is a particularlyuseful biomarker in identifying immunosuppressive regulatory T cells,including particularly, FoxP3+ regulatory T-cells, in biologicalsamples. Accordingly, in a first aspect, the invention provides methodsof identifying whether a regulatory T-cell is an immunosuppressiveregulatory T-cell by determining the level of expression of CD127 by thecell. In some embodiments, the invention provides methods of determiningwhether a regulatory T-cell in an enriched T-cell sample is highlylikely to be a FoxP3+ immunosuppressive T regulatory cell by detectingonly one common determinant or cell surface antigen of the T-cell,CD127. In other embodiments, the invention provides methods ofidentifying immunosuppressive regulatory T-cells by identifyingCD4+cn12?1^(◯1)− T cells according to their level of expression of theCD4 and CD127 biomarkers in a biological sample. In some embodiments,the invention provides methods of identifying immunosuppressiveregulatory T-cells in a sample by identifying CD8+cn12?1^(◯1)− T cellsaccording to their level of expression of the CD8 and CD127 biomarkers.In some embodiments, the invention provides methods of selecting orisolating the cells so identified. In some embodiments, the samplescomprise T cells and are obtained from blood (e.g., isolated from PBMC),lymphoid, thymus or any specific tissues/organ sample of interest. Thesetissues or organs would include the pancreas, eye, heart, liver, nerves,intestine, skin, muscle, and joints.

In a second aspect, the invention, provides methods of making anisolated population of an immunosuppressive regulatory T-cells which aresubstantially FoxP3+ or include FoxP3+ T-cells. In one embodiment, thepopulation is made by obtaining a biological sample comprisingregulatory T-cells including CD12?1^(◯1)− and CD127+ cells anddetermining the level of expression of CD4 and/or the CD8 and CD127, onthe surface of the T-cells and isolating the immunosuppressiveCD4+CD127Lo/−, CD4+CD8+CD12?1^(◯1)− or CD8+CD12?1^(◯1)− T-cells fromthose T-cells which are CD127+_ In some embodiments, the isolatedpopulation is obtained by providing an enriched T-cell sample anddetermining the level of expression of only one common determinant,CD127, on cells in the enriched sample and isolating CD12?1^(◯1)− cellsfrom the enriched T-cell sample. In some preferred embodiments, theisolated population is substantially CD8+CD12?1^(◯1)− T cells and/orCD4+CD12?1^(◯1)− T cells. In some additional embodiments, the isolatedpopulation is substantially CD8+CD12?1^(◯1)− T cells and/orCD4+CD12?1^(◯1)− T cells which are FoxP3+ cells. The cells bearing themarkers can be isolated, for instance, by the use of labeled antibodiesor ligands with FACS or magnetic particles/bead technologies as known toone of ordinary skill in the art. Accordingly, in some embodiments, theinvention provides a method of identifying immunosuppressive regulatoryT-cells in a sample by screening T-cells in said sample to detectCD12?1^(◯1)− T-cells; and then identifying said detected CD12?1^(◯1)−T-cells as immunosuppressive regulatory T-cells. In some furtherembodiments, the identified immunosuppressive regulatory T-cells areisolated from the CD127+ cells. In some embodiments, the isolatedidentified cells taken together provide a population ofimmunosuppressive regulatory T-cells for use according to the invention.

In a third aspect, the invention provides methods of making expandedpopulations of isolated immunosuppressive regulatory T-cell populationswhich are substantially FoxP3+ by expanding an isolated T-cellpopulation obtained by the above methods. In some embodiments, theisolated FoxP3+ T cell population is expanded by contacting the isolatedT-cells of the population with antigen, alloantigen, oranti-CD3/anti-CD28 antibodies in the presence of IL-2 plus TGFbeta orrapamycin. Alternatively, in this aspect, the T-cells in the biologicalsample can first be expanded and then isolated by the above methods. Insome preferred embodiment, the isolated expanded population issubstantially CD8+CD12?1^(◯1)− T cells and/or CD4+CD12?1^(◯1)− T cellswhich are FoxP3+ cells.

In yet another aspect the invention provides methods of modulating animmune response in a subject, wherein the isolated or expanded, isolatedimmunosuppressive regulatory T-cell populations obtained by the abovemethods are administered to the subject.

In still another aspect, the invention provides, pharmaceuticalcompositions comprising isolated or expanded immunosuppressive FoxP3+T-cell populations obtained according to the above methods.

In another aspect, the invention provides methods for producing anantigen-specific or non-specific regulatory T cell enriched composition,and resultant compositions and methods of use. In one embodiment, theinvention provides a method of modulating an immune reaction in asubject, said method comprising (a) obtaining a population of antigenspecific or non-specific subject-compatible cells including CD4+ andCD4⋅cells and CD127+ and CD127− cells and CD25+ and CD2s− cells; (b)producing an antigen-specific or non-specific regulatory CD12?1^(◯1)− orFoxP3+ T cell enriched composition from said population of cells bysorting the cells on the basis of their levels of expression of theCD127 antigen and the CD4 antigen or the CD127 antigen and the CD8antigen, or the CD4, CD8, and CD127 antigens and (c) introducing saidcomposition into said subject to modulate said immune reaction in saidsubject. In some preferred embodiments, the immune response is anautoimmune response and the antigen is an autoantigen. In preferredembodiments, the cells are not sorted by their levels of expression ofthe CD25 common determinant.

In other aspects, the selection of the regulatory T-cell according tothe level of expression of common determinant 127 comprises selectionsbased upon one or more additional common determinants (e.g., CD4, CD8,and CD25) under the CD system (CD stands for cluster of designation) forclassifying monoclonal antibodies and their specific antigens wasestablished at the first workshop on leukocyte differentiation antigens(Paris: 1982), where the rule was also introduced that at least two mAbsthat recognize the same molecule are required in order to assign a newCD. It was further recommended that the term CD be used for theclustered antibodies and the term CD molecule or CD antigen be used forantigens. Accordingly, the invention also provides methods for producingan antigen-specific regulatory T cell enriched composition, andresultant compositions and methods of use. In one embodiment, forinstance, the invention provides a method of modulating an immunereaction in a subject, said method comprising (a) obtaining a populationof subject-compatible cells containing CD4+ and CD4⋅cells and CD127+ andCD127− cells and CD25+ and CD2S− cells; (b) producing anantigen-specific regulatory CD4+cn12?1^(◯1)− T cell enriched compositionfrom said population of cells by identifying and isolating cellsaccording to their expression of both CD4, CD25 and CD127 and (c)introducing said composition into said subject to modulate said immunereaction in said subject. In some embodiments, CD4+CDI2?1^(◯1). T cellenriched composition is substantially CD25+ enriched population obtainedby also identifying and isolating the regulatory T cells according totheir level of expression of CD25 wherein CD25+ and CD25hi cells areboth positively selected for. The identifying and isolating can proceedin any order or simultaneously by the use, for instance, of multiple,distinguishable fluorophores as labels on the antibodies used to detectthe common determinants.

In another embodiment, for instance, the invention provides a method ofmodulating an immune reaction in a subject, said method comprising (a)obtaining a population of subject-compatible cells containing CD127+ andCD127− cells and CD25hi/+ and CD2S− cells; (b) producing anantigen-specific regulatory CD25hi/+ locn12?1^(◯1)− orCD25hi/+CDI2?1^(◯1)− or CD25hiCDI2?1^(◯1)⋅T cell enriched compositionfrom said population of cells by identifying and isolating cellsaccording to their expression of both CD25 and CD127 and (c) introducingsaid composition into said subject to modulate said immune reaction insaid subject.

In still a further aspect, the invention provides kits comprisingmaterials useful in performing the above methods. A kit for isolating animmunosuppressive T-cell population, for instance, can comprise a ligandor antibody which binds CD127. The ligand or antibody is preferablylabeled (e.g., a fluorescent or colored label or magnetic bead label) ora label to detect a cell bound by the ligand or antibody is providedwith the kit (e.g., fluorescently labeled or magnetically labeledantibody which binds to the CD127 antibody). In some embodiments, thekit would further provide instructions for obtaining animmunosuppressive T-cell population by use of the kits contents andpositive selection of T-reg cells which are low and/or negative CD127expressers. In some embodiments, the kit instructions would set forthCD127 as a selection marker along with CD4 and/or CD25 as positiveselection markers. In some embodiments, the kit provides a single CD127antibody for use in selecting for T-reg cells which are low and/ornegative CD127 expressers. In some embodiments, monoclonal antibodiesfor each of CD127 and CD4 and also optionally CD25 would be provided inthe kit. These antibodies may be labeled (e.g., attached to afluorescent label or magnetic label). In addition, the kit of couldfurther provide instructions for obtaining a T-cell population for usein suppressing an immune response and optionally, further, instructionsfor formulating the obtained population in a suitable media forcontacting with cells in vivo or in vitro. In some embodiments, of anyof the above, the antibodies in the kit are not labeled, but the kitadditionally provides means for labeling the antibodies (e.g., labeledantibodies which bind to an antibody used to bind CD127). In somefurther embodiments of any of the above, there is a proviso that the kitdoes not contain a means or antibody for detecting CD25. In someembodiments, the isolated population of cells, has not been directlyselected against having CD2S− and/or CD25¹⁰ cells. In some furtherembodiments, of any of the above, the kit provides further instructionfor separating the cells using a FACS (fluorescence activated cellsorter) in the case where a fluorescent label is used or magnetism inthe case where magnetic particles or beads are used as the label.

In still another aspect, the invention provides methods for identifyingimmunosuppressive drugs by determining their effect on only one T-cellsurface marker, CD127, or on a plurality of markers including CD127 andoptionally one or the other or both of CD4 and CD25. In some preferredembodiments, the assay is conducted using Tregs from an enriched T-cellsample.

With regard to any of the above aspects having embodiments whereinselection on the basis of the expression level of CD25 is not excludedsubject matter, in preferred embodiments of such the selection of theregulatory T-cell positively selects cells which are CD25+ or CD25¹⁰ inaddition to cells which are CD25hi_ With regard to any of the aboveaspects having embodiments wherein selection on the basis of theexpression level of CD25 is not excluded subject matter, in otherembodiments of such selection on the basis of CD25 expression levels isexcluded.

With regard to any of the above aspects having embodiments wherein stepsare provided, the steps may be performed in any order, sequentially orcontemporaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . FoxP3 is expressed on a significant percentage of CD4+ T cellsindependent of CD25 expression. Human PBMCs were cell surface stainedusing a combination of anti-CD4 and anti-CD25 mAbs. Once fixed, thecells were stained additionally with anti-FoxP3 mAb. Data arerepresentative of greater than 20 independent individuals and more thanIO experiments. The numbers in the dot plot indicate the percentage ofFoxP3+ cells.

FIG. 2A-2B. Expression of FoxP3 on different CD4+CD127+/− human T cellsubsets. FIG. 2A: PBMCs were harvested from human peripheral blood andstained with CD4, CD25, and CD127 and analyzed on a Becton-Dickenson LSRII. and intracellularly with FoxP3-specific mAbs. Data arerepresentative of greater than 20 independent individuals and more thanI0 experiments. FIG. 2B: Human PBMCs were stained for cell surfaceexpression of CD4 and CD127. The stained cells were fixed and stainedintracellularly for FoxP3. For analysis, the PBMCs were gated onlymphocytes (based on forward and side light scatter) and analyzed forCD127 and FoxP3 expression. The numbers in the dot plot indicate thepercentage of gated cells expressing the relevant marker. Data arerepresentative of greater than 20 independent individuals and more thanIO experiments.

FIG. 3A-3B. Expression of FoxP3 on different CD4+CD127+/− mouse T cellsubsets. FIG. 3A: Mouse spleen and lymph node cells were stained forcell surface expression of CD4 and CD127. For analysis, the spleen cellsfrom FoxP3-GFP mice were gated on lymphocytes (based on forward and sidelight scatter) and analyzed for CD127 and FoxP3 (GFP) expression. Thenumbers in the dot plot indicate the percentage of gated cellsexpressing the relevant marker. FIG. 3B: Spleen and lymph node cellsisolated from FoxP3 transgenic mice were stained for cell surfaceexpression of CD4, CD25 and CD127. For analysis, the spleen cells weregated on CD4+ lymphocytes (based on forward and side light scatter) andanalyzed for CD127 and FoxP3 expression. The numbers in the dot plotindicate the percentage of gated cells expressing the relevant marker.

FIG. 4A-4C. Expression of FoxP3 on different CD4+ T cell subsets. FIG.4A: Human PBMCs were stained for cell surface expression of CD4 andCD127. The stained cells were fixed and stained intracellularly forFoxP3. For analysis, the PBMCs were gated on CD4+ lymphocytes (based onforward and side light scatter and CD4 staining) and analyzed for CD127and FoxP3 expression. The boxes represent arbitrary designations ofCD25+ versus CD25− cells. The numbers in the dot plot indicate thepercentage of gated cells expressing the relevant marker. FIG. 4B: HumanPBMCs were stained for cell surface expression of CD4 and CD127. Thestained cells were fixed and stained intracellularly for FoxP3. Foranalysis, the PBMCs were gated on lymphocytes (based on forward and sidelight scatter) and analyzed for CD4, CD127 and FoxP3 expression. Theboxes represent arbitrary designations of CD127+ versus CDI2?1^(◯1)−cells. The numbers in the dot plot indicate the percentage of gatedcells expressing the relevant marker. FIG. 4C: Similar staining andanalysis was performed on whole blood obtained from I 0healthyindividuals. Each symbol represents an individual person, the narrow barrepresents the mean percentage of FoxP3+ T cells on either CD4+ T cellsgated based on CD25 and/or CD127 expression.

FIG. 5A-5B. ChIP-CHIP and ChIP-qPCR analysis of FoxP3 bound DNA fromCD4+CD25hi human Tregs. Anti-FoxP3 or control rabbit lg was used toprecipitate cross-linked protein-DNA complexes from expanded CD4+CD25hihuman Tregs lysate. The cross-linking of the immunoprecipitated materialwas removed, protease-treated, and the DNA was purified and amplified.The resultant material was hybridized to the whole genome usingGeneChip® Human tiling I.OR array set to identify the locations ofbinding sites for FoxP3. 2 sets of graphs: FoxP3 IP vs the lg controland FoxP3 IP vs Input DNA were generated on the hs.NCBiv35 version ofthe genome essentially following the method described in Cawley et el.(50). FIG. 5A: Signal enrichment graphs of IL-7R locus(chrS:35863179-35918811). Several regions in IL-7R locus are predictedto be positive (chrS:35892564-35892809 promoter) and negative(chrS:35890618-35890846 2K upstream; chrS:35907667-35907852 Intron 4;chrS:35911721-35911888 intron 7 and exon 8). FIG. 5B: SYBR green qPCR ofIL-7R chromosomal regions. FoxP3 IP vs the IgG fold enrichment ratio wasdetermined from duplicate ChIP assay evaluated in duplicate by real timePCR.

FIG. 6 . Proliferative response of isolated T cell subsets. Buffy coatsamples were sorted based on CD4, CD127 and CD25 expression. 30,000sorted cells were put into culture with allogeneic anti-CD3-depleted,irradiated, third party PBMC as stimulators. T cells were incubated for7 days at 37° C. in 5% CO2. Sixteen hours before the end of theincubation, I μCi³H-thymidine was added to each well. Plates wereharvested and data analyzed. Data is representative of 9 separateexperiments.

FIG. 7A-7B. Suppression of allogeneic MLR by individual T cell subsets.Buffy coat samples were sorted based on CD127 and CD25 expression.30,000 sorted cells were combined with 100,000 autologous PBMC asresponders, and 100,000 allogeneic anti-CD3-depleted, irradiated thirdparty PBMC as stimulators. T cells were incubated for 7 days at 37° C.in 5% CO2. Sixteen hours before the end of the incubation, 1 μCi³H-thymidine was added to each well. Plates were harvested and dataanalyzed. Data is representative of 9 separate experiments sorting 7different subpopulations of CD4+ cells indicated. FIG. 7A: CD127+CD25+,CD127+CD2s⋅, CD12?1^(◯1) ⋅CD25+, CD12?1^(◯1)⋅CD2S− and FIG. 7B:CD12?1^(◯1)⋅, CD25m, CD127+. 100,000 responders are present in each wellwith decreasing numbers of sorted cells added at 1:1 ratio(30,000:100,000), 1:1/2 (15,000 sorted cells), 1:1/4 (7,500 sortedcells), 1:1/16 (1,875 sorted cells) in comparison to sorted cells alone.Results are represented as counts per minute (CPM).

FIG. 8 . Frequency of various T cell subsets in patients with Type 1Diabetes versus healthy control subjects. The data was obtained fromtotal 10 healthy control individuals and 16 patients with Type 1Diabetes. The mean percentage of various T cell subsets and standarddeviation.

FIG. 9 . An amino acid sequence of a human CD127 protein (SEQ ID NO:1).

FIG. 10 . An amino acid sequence of a human CD4 protein (SEQ ID NO:2).

FIG. 11 . FoxP3 expression on day 14 of expanded CD4+CD127− T cells.Separated CD4+CD12?1^(◯) cells were cultured for 14 days withanti-CD3/anti-CD28 plus IL-2 and plus or minus rapamycin (RAPA). As canbe seen the cells expanded best in the absence of rapamycin and whenrestimulated with the mAb and IL-2 cocktail on day 9. Extent ofexpansion shown in right hand boxes. The cell expanded with RAPA had thehighest levels of FoxP3 and percentage of FoxP3+ cells. The majority ofFoxP3 negative cells were CD25¹⁰ versus FoxP3+ cells in the sameculture.

FIG. 12 . Expanded CD4+CD12?1^(◯1)⋅Tregs suppress and maintain FoxP3,especially when treated with rapamycin. At 14 days, expanded cells wereseparated and added to a CFSE suppression assay.

FIG. 13A-13B. Expanded CD4+cn12?1^(◯1)− Tregs suppress and maintainFoxP3, especially when treated with rapamycin. Comparison ofCD4+CD2?1^(◯) suppression based on CD25 separation after expansion. At14 days, expanded cells were separated into CD25+(middle column) andCD2S− (right column) subsets and added to a CFSE suppression assay.Interestingly, after culture both populations suppressed equivalentlysuggesting that CD25 expression was not essential to confer suppressiveactivity on the expanded cells. However, we were unable to rule outwhether the cells that suppressed in these cultures were derived fromCD25+ cells or had down-regulated CD25 during the culture.

FIG. 14A-14B. FoxP3 and CD127 expression (FIG. 14A) and Treg function(FIG. 14B) of fresh CD4+CD12?1^(◯) FoxP3+ and FoxP3− mouse T cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

By a “population of cells” is meant a plurality of cells, preferably atleast 10³, 104, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ cells. Thepopulation in some embodiments has from 10⁵ to 10⁷ cells, 10⁶ to 10⁸cells, or from 10⁸ to 10¹¹ cells, or 10¹⁰ to 10¹² cells.

A therapeutically effective amount of isolated or expanded isolatedCD127Lo/− regulatory T-cells provided by the invention is generallybetween 10⁷ to 10¹¹ cells, and more preferably 10⁷ to 10⁹ cells.

Immune conditions, diseases, disorders and reactions or responses to betreated according to the methods and compositions of the invention meansa disease in which the immune system contributes to pathogenesis. Thesereactions include, but are not limited to, autoimmune conditions,disorders or diseases and persistent and progressive immune reactions toinfectious non self antigens from bacterial, viral (e.g., HCV), fungal,or parasitic organisms which invade and persist within mammals andhumans. Such conditions and disorders include allergies and/or asthma.The allergies and asthma may be due to sensitization with foreign ornon-self antigens as pollen, animal dander and food proteins. The sourceof the provoking foreign antigen can be plant, fungal, mold, or otherenvironmental contaminant.

Autoimmunity and Autoimmune Disorders and Diseases. Autoimmunity isdefined as persistent and progressive immune reactions to non infectiousself antigens, as distinct from infectious non self antigens frombacterial, viral, fungal, or parasitic organisms which invade andpersist within mammals and humans. Autoimmune conditions includescleroderma, Grave's disease, Crohn's disease, Sjorgen's disease,multiple sclerosis, Hashimoto's disease, psoriasis, myasathenia gravis,Autoimmune Polyendocrinopathy syndromes, Type I diabetes mellitus(TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis,colitis, and thyroiditis, as well as in the generalized autoimmunediseases typified by human Lupus. “Autoantigen” or “self-antigen” asused herein refers to an antigen or epitope which is native to themammal and which is immunogenic in said mammal disease.

A patient with an autoimmune disease may be diagnosed as known to one ofordinary skill in the art. Such patients may be identifiedsymptomatically and/or by obtaining a sample from a patient andisolating autoreactive T cells and comparing the level of autoreactive Tcells in a patient to a control (see, U.S. Patent ApplicationPublication No. 20060105336). For instance, type 1 diabetes may beidentified by age of on-set and dependence on insulin injections tomaintain glucose homeostasis.

The response of a patient with an autoimmune disease to treatment may bemonitored by determining the severity of their symptoms or bydetermining the frequency of autoreactive T cells in a sample from apatient with an autoimmune disease. The severity of symptoms of theautoimmune disease may correlate with the number of autoreactive T cells(see, U.S. Patent Application Publication No. 20060105336). In addition,an increase in the number of autoreactive T cells in the sample may beused as an indication to apply treatments intended to minimize theseverity of the symptoms and/or treat the disease before the symptomsappear.

“CD,” “cluster of differentiation” or “common determinant” as usedherein refers to cell surface molecules recognized by antibodies.Expression of some CDs (e.g., CD4, CD8, CD25, CD127) is specific forcells of a particular lineage or maturational pathway, and theexpression of others varies according to the state of activation,position, or differentiation of the same cells. Preferably, in someembodiments, the CD determinants are human when the isolated cells areto be administered to a human or a human immune response is beingstudied.

As used herein, the term “CD127” refers to the “interleukin-7 receptor,”present on a Treg cell surface. The IL-7 receptor alpha chain isdescribed in the literature. See, e.g., Goodwin et al. (1990) Cell60:941-951; GenBank Accession Nos. NP.sub.-032398 and NP.sub.-002176.IL-7R is also referred to in the literature as CD127. (see FIG. 9 ). Theterm CD127 ligand refers to a compound that binds to the IL-7 receptor.(see, U.S. Pat. Nos. 5,194,375 and 5,264,416). CD127+ refers to cellswhich stain intensely or brightly when treated with a labeled antibodydirected toward CD127. CD127Lo/− refers to cells of a type which stainsslightly/dully or not at all when contacted with a labeled CD127antibody. Generally, the cells are distinguished according to theirCD127 expression levels based upon a readily discernible differences instaining intensity as is known to one of ordinary skill in the art. Insome embodiments, the cut off for designating a cell as a CD127Lo/− cellcan be set in terms of the fluorescent intensity distribution observedfor all the cells with those cells falling below the 50%, 40%, 30% or20% of fluoresecence intensity being designated as CD127Lo/− cells. ACD127− cell can be designated as one which falls below the tenth bottompercentile with respect to fluorescence intensity. In some embodiments,the frequency distribution of the CD127 staining is obtained for all thecells and the population curve fit to a higher staining and lowerstaining population, and cells assigned to the population to which theymost statistically are likely to belong in view of a statisticalanalysis of the respective population distributions. In someembodiments, the CD12?1^(◯1)− cells stain two to three fold lessintensely than the CD127+ cells. Particularly preferred methods are alsoexemplified in the Examples.

As used herein, the term “CD4” refers to a cell-surface glycoproteintypically found on the mature helper T cells and immature thymocytes, aswell as on monocytes and macrophages. On T cells, CD4 is the co-receptorfor the T cell receptor (TCR) and recruits the tyrosine kinase Ick. Withits DI-portion, CD4 can attach to the 2-domain of MHC class IImolecules. CD4+ refers to cells which stain brightly when contacted withlabeled anti-CD4 antibody, and CD4 refers to cells of a type which stainthe least brightly, dull or not at all, when contacted with afluorescently labeled CD4 antibody. Generally, the cells aredistinguished according to their CD4 expression levels based upon areadily discernible differences in staining intensity as the CD4staining is clearly bimodal. In some embodiments, the frequencydistribution of the CD4 staining is obtained for all the cells and thepopulation curve fit to a higher staining and lower staining population,and cells assigned to the population to which they most statisticallyare likely to belong in view of a statistical analysis of the respectivepopulation distributions. In some embodiments, the CD4 cells stain twoto three fold less intensely than the CD4+ cells. Particularly preferredmethods are also exemplified in the Examples.

Methods of segregating CD8 T cells into + and − categories are known topersons of ordinary skill in the art. In some embodiments, the frequencydistribution of the CD8 staining is obtained for all the cells and thepopulation curve fit to a higher staining and lower staining population,and cells assigned to the population to which they most statisticallyare likely to belong in view of a statistical analysis of the respectivepopulation distributions. In some embodiments, the CD8+ cells stain twoto three fold more intensely than the CDS cells. Particularly preferredmethods are also exemplified in the Examples.

As used herein, the term “CD25” refers to the alpha subunit ofinterleukin-2 receptor, a single-chain glycoprotein with a molecularweight of 55 kD. Following the activation of T cells with antigen ormitogen in the presence of the monokine interleukin-I, interleukin 2(IL-2) is rapidly synthesised and secreted. In response to this, asubpopulation of T cells expresses high affinity receptors for IL-2.These cells proliferate, expanding the T cell population which iscapable of mediating helper, suppressor and cytotoxic functions. IL-2receptor is not uniquely found on T cells. CD25hi refers to cells whichstain brightly when contacted with labeled anti-CD25 antibody, CD25+refers to cells which stain less brightly when contacted with labeledanti-CD25 antibody, and CD25¹⁰¹ refers to cells which are of a typewhich stains the least brightly dull or null when contacted with alabeled CD25 antibody. Generally, the cells are distinguished accordingto their CD25 expression levels based upon differences in stainingintensity as is known to one of ordinary skill in the art. In someembodiments, the cut off for designating a cell as a CD25 expressioncategory hi, +, lo, or − cell can be set in terms of the fluorescentintensity distribution observed for all the cells. Generally, cells inthe top 2, 3, 4, or 5% of staining intensity are designated “hi”, withthose falling in the top half of the population categorized as being“+”. Those cells falling below 50%, of fluoresecence intensity aredesignated as CD25¹⁰ cells and below 5% as CD2S− cells. Suitable methodsof identifying and categorizing regulatory T-cells with respect to theirexpression of CD25 are described further in Baecher-Allen (41), which isincorporated by reference with respect to same. Particularly preferredmethods are also exemplified in the Examples.

A CD12?1^(◯1)⋅cell accordingly is of a type which stains the leastbrightly, dull or null when contacted with a labeled CD127 antibody.Most preferably, the CD12?1^(◯1)-cells are FoxP3+ cells. In someembodiments the CD12?1^(◯1)-cells are CD127⋅cells,CD4+CD12?1^(◯1)⋅cells, CD25+CD4+CD127− cells, CD2S−CD4+CD127− cells,CD2sm− CD4+CD127− cells⋅ or CD2sm−CD4+CD12?1^(◯1)− cells. Thedesignation of a cell type with respect to its levels of expression of arecited biomarker or CD is meant to describe the cell being referencedby its biomarker expression phenotype and is not necessarily anindicator that expression levels were actually determined for thereferenced cell. In preferred embodiments, the CD expression pattern ofthe CD12?1^(◯1) cell was determined only with respect to CD127.

CD12?1^(◯1)-regulatory T-cell populations for use according to theinvention are cell populations which have been negatively selected forthe CD127 biomarker. In some embodiments, the cells have been furthercharacterized with respect to other CD determinants, particularly theCD4, CD8 and CD25 determinants (e.g., positively selected for withrespect to CD4, CD8 and/or CD25). In other embodiments, the cells havebeen further characterized according to their expression of commondeterminants other than CD4 and/or CD25. In preferred embodiments, thecell populations are substantially the selected cell type.

In some embodiments, the immunosuppressive T-cell inhibits theproduction of IL-2 or the proliferation of T-cells in an assay (e.g.,MLR). These and other methods of assaying T cells for immunosuppressiveactivity are known to persons of ordinary skill in the art.

As used herein, the term “sample” or “biological sample” refers totissues or body fluids removed from a mammal, preferably human, andwhich contain regulatory T cells, including, but not limited to, FoxP3+T cells and/or CD12?1^(◯1)− regulatory T-cells. In some embodiments, thesamples are taken from individuals with an immune response which needsto be suppressed. In some embodiments, the individual has an allergy,Graft vs. Host Disease, an organ transplant, or autoimmune disorder.Samples preferably are blood and blood fractions, including peripheralblood. The biological sample is drawn from the body of a mammal, such asa human, and may be blood, bone marrow cells, or similar tissues orcells from an organ afflicted with the unwanted immune response. Methodsfor obtaining such samples are well known to workers in the fields ofcellular immunology and surgery. They include sampling blood in wellknown ways, or obtaining biopsies from the bone marrow or other tissueor organ. In preferred embodiments, the sample is a T-cell enrichedsample in which the sample cells are substantially T-cells.

Enriched T-cell samples refer to those samples or biological samplesthat have been enriched for T cells by positive selection of the T cellsbearing the CD4 marker and/or positive selection of the CD8 marker bydetermining the levels of expression of the CD4 and CD8 markers,respectively. Other enriched T-cell samples have been enriched forT-cells by negative selection of (i.e., selecting against) non-T-cellswhich can be distinguished by their levels of expression of other commondeterminants exclusive of CD25. Most preferably, the enrichment of thesample for T-cells is not specifically according to their level ofexpression of CD25. Accordingly, in preferred embodiments, the enrichedsample substantially comprises a regulatory T-cell population whichcomprises at least 2, 4, 6, 8, 10, 12, or 20% CD25+ cells or CD25¹⁰ orCD25⋅cells.

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibiting, activating, or modulatingcells, respectively, and are identified using in vitro and in vivoassays for expression or activity. The term “modulator” includesinhibitors and activators. A modulator can be an antibody or a solubleligand which binds a protein of interest. Inhibitors are agents that,e.g., inhibit expression of a polypeptide or polynucleotide of theinvention or bind to, partially or totally block stimulation orenzymatic activity, decrease, prevent, delay activation, inactivate,desensitize, or down regulate the activity of a polypeptide orpolynucleotide of the invention, e.g., antagonists. Preferred modulatorsaccording to the invention, inhibit or suppress immune responses to anantigen or alloantigen. Assays to identify inhibitors and activatorsinclude, e.g., applying putative modulators to immune cells and thendetermining the functional effects of the cell on the immune response(e.g., MLR). Inhibitors or modulators are compared to control sampleswithout the inhibitor or modulator to examine the extent of effect.Control samples (untreated with modulators) are assigned a relativeactivity value of 100%. Inhibition is achieved when the activity valueof a polypeptide or polynucleotide of the invention relative to thecontrol sample is about 80%, optionally 50% or 25 to 1%, or less.Activation is achieved when the activity value of a polypeptide orpolynucleotide of the invention relative to the control sample is 110%,optionally 150%, optionally 200-500%, or 1000-3000%, or higher.

The term “isolated” with regard to a population of cells as used hereinrefers to a cell population which either has no naturally-occurringcounterpart or has been separated or purified from other components,including other cell types, which naturally accompany it, e.g., innormal or diseased tissues such as lung, kidney, or placenta, tumortissue such as colon cancer tissue, or body fluids such as blood, serum,or urine. Typically, an isolated cell population is at least two fold,four-fold, or eight-fold enriched for a specified cell type whencompared to the natural source from which the population was obtained.

A population or subpopulation of cells which is “substantially” of aspecified cell type is one which has a count of the specified cell typewhich is at least 50%, 75%, 80%, 90%, 95% or, most preferably, 98% or99% of the total cell count of the population or subpopulation or onewhich is at least two-fold, four-fold, eight-fold, ten-fold or 20-foldenriched for a specified cell type as compared to a source population ofthe specified cell type.

An “anti-X antibody” or “X antibody” according to the invention is anantibody which can specifically bind to X. For instance, the anti-CD127antibody or CD127 antibody is capable of binding CD127. The antibodiesfor use according to the invention include, but are not limited to,recombinant antibodies, polyclonal antibodies, monoclonal antibodies,chimeric antibodies, human monoclonal antibodies, humanized orprimatized monoclonal antibodies, and antibody fragments. A great manylymphocyte biomarker specific antibodies are commercially available.These include anti-CD127, anti-CD4, and anti-CD25 antibodies.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de nova either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de nova using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)). In some embodiments, a high affinity ligand of a target may beused in place of the antibody.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Specific binding to an antibody under suchconditions requires an antibody that is selected for its specificity fora particular protein. For example, polyclonal antibodies can be selectedto obtain only those polyclonal antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules.

Preferably a “label” or a “detectable moiety” is covalently ornoncovalently attached to the antibody. A label may be detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. Particularly useful labels are fluorescent dyes.Methods of attaching labels to antibodies are well known to those ofordinary skill in the art. Particularly preferred labels are those whichare attached to the antibody by a linker which can be readily cleaved orseparated or subject to hydrolysis by contact with a predeterminedenzyme under physiological conditions. The antibody may also beconjugated with a magnetic particle, such as a paramagnetic microbead(Miltenyi Biotec, Germany). An activated T cell bound by a magneticallylabeled antibody may be isolated using techniques including, but notlimited to, magnetic cell sorting. Suitably labeled antibodies to CD127,CD4 and CD25, as well as many other CDs, are commercially available andknown to one of ordinary skill in the art. The antibody may be labeledbefore or after contact with the sample or before or after contact withthe CD. The CD antibody may be labeled by contacting with an a labeledantibody which binds to the CD-antibody.

The term “test compound” or “candidate molecule” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, polypeptide,oligopeptide (e.g., from about 5 to about 25 amino acids in length,preferably from about 10 to 20 or 12 to 18 amino acids in length,preferably 12, 15, or 18 amino acids in length), small organic molecule,polysaccharide, lipid, fatty acid, polynucleotide, RNAi,oligonucleotide, etc. The test compound can be in the form of a libraryof test compounds, such as a combinatorial or randomized library thatprovides a sufficient range of diversity. Test compounds are optionallylinked to a fusion partner, e.g., targeting compounds, rescue compounds,dimerization compounds, stabilizing compounds, addressable compounds,and other functional moieties. Conventionally, new chemical entitieswith useful immunosuppressive properties are generated by identifying atest compound (called a “lead compound”) with some desirable property oractivity, e.g., inhibiting expression of CD127, upregulating expressionof FoxP3, and creating variants of the lead compound, and evaluating theproperty and activity of those variant compounds. Often, high throughputscreening (HTS) methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, preferably lessthan about 2000 Daltons, preferably between about 100 to about 1000Daltons, more preferably between about 200 to about 500 Daltons.

“Determining the functional effect” refers to assaying for a compoundthat increases or decreases the expression of CD127. Such functionaleffects can be measured by any means known to those skilled in the art,e.g., changes in spectroscopic (e.g., fluorescence, absorbance,refractive index), hydrodynamic (e.g., shape), chromatographic, orsolubility properties for the protein; measuring inducible markers ortranscriptional activation of the protein; measuring binding activity orbinding assays, e.g. binding to antibodies; measuring changes in ligandbinding affinity; e.g., via chemiluminescence, fluorescence,colorimetric reactions, antibody binding, inducible markers, and ligandbinding assays.

Samples or assays for identifying immunosuppressive agents or drugs areconducted in the presence of the candidate inhibitor of CD127 expressionand then the results are compared to control samples without theinhibitor to examine for the desired activity or to determine thefunctional effect of the candidate inhibitor. A positive referencecontrol which is an agent having the desired activity may be used.Control samples (untreated with inhibitors) are assigned a relative of100%. Inhibition is achieved when the activity value relative to thecontrol is about 80%, preferably 50%, more preferably 25 to 1%, or evenless (e.g., 0.2%, 0%).

Discoveries and Findings

We have discovered that CD127, even in the absence of CD25 selection, isan excellent marker of Tregs in human peripheral blood. The cell surfacemarker is expressed at low levels on an overwhelming majority of Tregsand distinguishes up to 20% of CD4+ T cells as potential Tregs.Moreover, the cell surface marker can be used, without determining theexpression of levels of CD25 on the regulatory T-cells, to separate asuppressive T cell subset and will thus be a useful tool for theselection and expansion of T cell for diagnostics and therapeuticapplications. Reliance upon the CD127 marker expression alone, has theadvantage of convenience and can afford a substantially greater yield ofFoxP3+ cells when compared to methods relying on, for instance positiveselection of both the CD4 and CD25 biomarkers.

As noted earlier, regulatory T cells (Treg) are critical regulators ofimmune tolerance. Most Treg are defined based on expression of CD4, CD25and the transcription factor, FoxP3. Recent information has identifiedthe importance of the gene Foxpro3 which is induced by thymus epitheliumto cause T cells to develop CD25+ CD4+ regulatory T cells, in animalmodels of autoimmune diseases. Deficiency of this gene may lead to widespread autoimmune phenomena and diseases. However, these markers haveproven problematic for uniquely defining this specialized T cell subsetin humans. We have found that IL-7 receptor (CD127) is down-regulated ona subset of CD4+ regulatory T cells in peripheral blood. We demonstratethat the majority of these cells are FoxP3+, including those thatexpress low levels or no CD25. A combination of CD4, CD25 and CD127resulted in a highly purified population of Tregs accounting forsignificantly more cells that previously identified based on other cellsurface markers. These cells were highly suppressive in functionalsuppressor assays. In fact, cells separated based solely on CD4 andCD127 expression were anergic and, although representing at least3-times the number of cells (including both CD25+CD4+ and CD2S-CD4+ Tcell subsets), were as suppressive as the “classic” CD4+CD25hi Tregsubset. Finally, we show that CD127 can be used to quantitate Tregsubsets in individuals with Type I diabetes supporting the use of CD127as a biomarker for human Treg.

In an effort to define new biomarkers of human Tregs, we have combinedgene expression microarray, flow cytometry and function assays toidentify new cell surface proteins that distinguish human Tregs. Weobserved that IL-7R (CD127) is downregulated on all human T cells afteractivation. In contrast to the reported re-expression of CD127 on themajority of effector and memory T cells (32-35), FoxP3+ T cells remainCDI2?1^(◯1)−. In fact, the CDI2?1^(◯1)−, FoxP3+ T cells accounted for asignificant percentage of CD4+ T cells in the peripheral blood. Wedemonstrate that FoxP3 interacts with the CD127 promoter and given itspurported repressor function likely contributes to the reducedexpression of CD127 in Tregs. Finally, we show that isolatedCD4+CDI2?1^(◯1)− T cell subset is anergic and suppresses alloantigenresponses in vitro. Together, these data suggest a dichotomy betweenmemory T cells which are IL-2R¹⁰IL-7Rhi and regulatory FoxP3+ T cellswhich in most instances up-regulate IL-2R while remaining IL-7R¹⁰¹,(30). Thus, the CD127 biomarker can be used to selectively enrich humanTregs for in vitro functional studies and in vivo therapy.

Embodiments

Accordingly, in a first aspect, the invention provides methods ofidentifying whether a regulatory T-cell is a suppressive regulatoryT-cell by detecting only one cell surface antigen of the T-cell, CD127in an enriched T cell sample. In some embodiments, the inventionprovides methods of determining whether a regulatory T-cell is highlylikely to be a FoxP3+ suppressive T regulatory cell by detecting onlyone cell surface antigen of the T-cell, CD127 in an enriched T-cellsample. In preferred embodiments, the CD12i⁰¹-cells are identified bycontacting regulatory T-cells in the sample with fluorescently-labeledmonoclonal antibodies which specifically bind to CD127+ and identifyingcells having a reduced level of fluorescent label attached thereto. TheCD12i⁰¹⋅T-cells in large proportion are immunosuppressive or FoxP3+cells. In further embodiments, the CD12i⁰¹⋅T-cells are CD127− T-cells.In further embodiments, the invention provides methods of estimating thenumber of FoxP3 positive regulatory T-cells in a sample by detectingonly one cell surface antigen of the T-cell, CD127. In otherembodiments, the invention provides methods of identifying regulatoryT-cells by identifying CD4+CD12i⁰¹⋅T cells according to their level ofexpression of the CD4 and CD127 biomarkers in a biological sample. Insome embodiments, the invention provides methods of identifyingregulatory T-cells in a sample by identifying CD8+CD12i⁰¹− T cellsaccording to their level of expression of the CD8 and CD127 biomarkers.

In a second aspect, the invention, provides methods of making anisolated population of an immunosuppressive regulatory T-cells which aresubstantially FoxP3+ by obtaining a biological sample comprisingregulatory T-cells including, but not limited to, CD12i⁰¹− and CD127+cells and determining the level of expression of CD127, on the surfaceof the T-cells and isolating the immunosuppressive CD127Lo/− or CD127−T-cells from those T-cells which are CD127+. In some embodiments, thesample is an enriched T-cell sample. In some embodiments, the T-cellpopulations so made have at least 4, 8, 10, or 20% CD25+ and¹ orCD2S-regulatory T cells. In some embodiments, the isolated population isobtained by providing an enriched T-cell sample and determining thelevel of expression of only one common determinant, CD127, on cells inthe enriched sample and isolating CD12i⁰¹− cells from the enrichedT-cell sample. In some preferred embodiment, the isolated population issubstantially CD8+CD12i⁰¹− T cells and/or CD4+CD12i⁰¹− T cells which areFox 3+ cells.

In a third aspect, the invention provides methods of making expandedpopulations of isolated immunosuppressive regulatory T-cell populationswhich are substantially FoxP3+ by expanding an isolated T-cellpopulation obtained by the above methods. In some embodiments, theisolated FoxP3+ T cell population is expanded by contacting the isolatedT-cells of the population with antigen, alloantigen, oranti-CD3/anti-CD28 antibodies in the presence of TGFbeta or rapamycin.Alternatively, in this aspect, the T-cells in the biological sample canfirst be expanded and then isolated by the above methods. In preferredembodiments of either approach, the expanded isolated immunosuppressiveT-cell populations comprise at least 2, 4, 8, 10, or 20% CD2s− and orCD25+ T regulatory cells. In preferred embodiments, the CD12i⁰¹-cellsare identified by contacting regulatory T-cells in the sample withfluorescently-labeled monoclonal antibodies which specifically bind toCD127+ and sorting the regulatory T-cells according to their level ofexpression of CD127 using a fluorescently activated cell sorter (FACS).In some preferred embodiment, the isolated expanded population issubstantially CD8+cn12i⁰¹− T cells and/or CD4+cn12i⁰¹− T cells which areFox p3+ cells.

In yet another aspect the invention provides methods of modulating animmune response in a subject, wherein the isolated or expanded, isolatedimmunosuppressive regulatory T-cell populations obtained by the abovemethods are administered to the subject. The biological sample fromwhich the regulatory T-cells are obtained may be autologous in that thebiological sample itself was obtained from the subject to be treated.The subject can be any mammal (e.g., human, primate) in which modulationof an immune reaction is desired. In some embodiments, a human ormammalian subject has immune disorder, disease or condition or anautoimmune disease to be treated. There are numerous, established animalmodels for using T cell epitopes of autoantigens to induce tolerance,including multiple sclerosis (EAE: experimental autoimmuneencephalomyelitis), myasthenia gravis (EMG: experimental myastheniagravis) and neuritis (EAN: experimental autoimmune neuritis). In anotherembodiment, the subject is a human afflicted with an autoimmune diseaseor disorder, such as any of the diseases/disorders listed in Table A.

In still another aspect, the invention provides, pharmaceuticalcompositions comprising isolated or expanded immunosuppressive FoxP3+T-cell populations obtained according to the above methods.

In another aspect, the invention provides methods for producing anantigen-specific regulatory T cell enriched composition, and resultantcompositions and methods of use. In one embodiment, the inventionprovides a method of modulating an immune reaction in a subject, saidmethod comprising (a) obtaining a population of subject-compatible cellsincluding CD4+ and CD4⋅cells and CD127+ and CD127− cells and CD25+ andCD2S− cells; (b) producing an antigen-specific regulatory CD12i⁰¹− orFoxP3+ T cell enriched composition from said population of cells bysorting the cells solely on the basis of the CD127 antigen, and (c)introducing said composition into said subject to modulate said immunereaction in said subject. In some preferred embodiments, the immuneresponse is an autoimmune response and the antigen is an autoantigen. Inother embodiments, the immune response is a graft vs. host immuneresponse and the antigen is an autoantigen. In other embodiments, theimmune response is allergy, asthma, tissue or organ transplantrejection, or a graft vs. host immune response and the antigen is apurified or unpurified component of the allergen or transplanted tissueor organ provoking the harmful immune response. In preferred embodimentsof any of the above, as the enriched composition was not directlyselected for on the basis of the presence or absence of CD25 biomarkeron the cells, the enriched composition comprises at least 2%, 4%, 6%,8%, 10% or 20% of CD2s− cells.

In yet further embodiments of any of the above aspects, the selection isnot based solely on the determining of the expression level of the CD127biomarker, but can include determining the expression levels ofsecondary T-cell surface antigen biomarkers or common determinants,including the CD4, and CD8 and CD25 biomarkers. In some such furthersuch embodiments, there is a proviso that no secondary T-cell surfaceantigen biomarker used isolating the cells is CD4. In still furtherother embodiments, there is a proviso that no secondary T-cell surfaceantigen used in isolating the cells is CD25. In some such furtherembodiments, there is a proviso that no secondary T-cell surface antigenbiomarker is CD4 or CD25. In further embodiments of any of the aboveaspects, the selection is not based solely on the expression level ofthe CD127 biomarker, but can include T-cell expression levels of theCD25 biomarker. In some other embodiments of any of the above aspects,the selection is not based solely on the expression level of the CD127biomarker, but can include T-cell expression levels of the CD4biomarker. In still some further embodiments of the above, the selectionis not based solely on the expression level of the CD127 biomarker, butcan include T-cell expression levels of both the CD4 biomarker and CD25biomarkers. Generally, in these embodiments, the CD4 and CD25 biomarkersare positively selected for and the CD127 biomarker is negativelyselected for. To enhance enrichment, positive selection may be combinedwith selection against cells comprising surface makers specific tonon-regulatory T-cell types, such as depletion of CD8, CD11b, CD16,CD19, CD36 and CD56-bearing cells as known to one or of ordinary skillin the art. Accordingly, the selection in any of the above embodiment bepracticed on an enriched T-cell sample.

In accordance with these further embodiments, the invention alsoprovides methods for producing an antigen-specific regulatory T cellenriched composition, and resultant compositions and methods of use. Inone embodiment, the invention provides a method of modulating an immunereaction in a subject, said method comprising (a) obtaining a populationof subject-compatible cells containing CD4+ and CD4− cells and CD127+and CD127− cells and CD25+ and CD2s− cells; (b) producing anantigen-specific regulatory CD4+CD12?1^(◯1)⋅T cell enriched compositionfrom said population of cells and (c) introducing said composition intosaid subject to modulate said immune reaction in said subject. In somepreferred embodiments, the immune response is an autoimmune response andthe antigen is an autoantigen. In other embodiments, the immune responseis a graft vs. host immune response and the antigen is an autoantigen.In other embodiments, the immune response is allergy, asthma, tissue ororgan transplant rejection, or a graft vs. host immune response and theantigen is a purified or unpurified component of the allergentransplanted tissue or organ provoking the immune response. In preferredembodiments of any of the above, the enriched composition was notdirectly selected for on the basis of the presence or absence orexpression of CD25 biomarker on the cells. In some embodiments,accordingly, the enriched composition comprises at least 2%, 4%, 6%, 8%,10% or 20% of CD2S− or CD25+ cells. In some embodiments, the enrichedcomposition was also enriched for CD25+ cells by sorting the cells onthe basis of the presence or absence of the CD25 biomarker on the cells.

In still a further aspect, the invention provides kits comprisingmaterials specifically useful in performing the above methods.

In still another aspect, the invention provides methods for identifyingimmunosuppressive drugs by determining their effect on only one T-cellsurface marker, CD127, or on a plurality of markers including CD127 andoptionally one or the other or both of CD4 and CD25. Drugs which areimmunosuppressive reduce the number of CD12?1^(◯1)− cells in a sampleT-cell population when contacted with a sample of such cells as comparedto a suitable control sample contacted with vehicle or anon-immunomodulatory substance.

In some embodiments, the invention further provides, an isolatedpopulation of regulatory T cells which are substantially CD4+ andCD12?1^(◯1)−. In other embodiments, the Treg cells were isolated from asample comprising the CD4+ and CD12?1^(◯1)− Treg cells and furthercomprising CD4− cells and CD127+ Treg cells by sorting the cells in thesample according to their expression of the CD4 biomarker and accordingto their expression of the CD127 biomarker. In some further embodiments,the isolated population is obtained by contacting a sample containingTreg cells with a labeled antibody specific for the CD4 biomarker andwith a labeled antibody specific for the CD127 biomarker to identifyTreg cells which are CD4+ and CD12?1^(◯1)−, and isolating the identifiedcells. In some embodiments, the CD4 antibody and the CD127 antibody areeach labeled with a different label. In further such embodiments, theCD4 antibody label and the CD127 antibody label are each a fluorescentlabel. In some further embodiments of any of the above, the sample isnot contacted with an antibody specific for the CD25 biomarker or forthe CD4 biomarker, or both. Preferably, the CD4+ and CD127¹⁰¹⋅cells areidentified and isolated in a fluorescence activated cell sorter. In someembodiments, of any of the above, the sample is a sample of peripheralblood. Preferably, in some embodiments, at least 2, 4, 8, 10, or 20% ofthe CD4+CD12?1^(◯1)− T-cells, of the population are also CD25¹⁰¹, orCD2s−. In other embodiments, the isolated population of Treg cells aresubstantially CD4+ and CD127−.

In some embodiments, the invention further provides, an isolatedpopulation of regulatory T cells which are substantially CD8+ andCD12?1^(◯1)−. In other embodiments, the Treg cells were isolated from asample comprising the CD8+ and CD12?1^(◯1)− Treg cells and furthercomprising ens⋅cells and CD127+ Treg cells by sorting the cells in thesample according to their expression of the CD8 biomarker and accordingto their expression of the CD127 biomarker. In some further embodiments,the isolated population is obtained by contacting a sample containingTreg cells with a labeled antibody specific for the CD8 biomarker andwith a labeled antibody specific for the CD127 biomarker to identifyTreg cells which are CD8+ and CD12?1^(◯1)−, and isolating the identifiedcells. In some embodiments, the CD8 antibody and the CD127 antibody areeach labeled with a different label. In further such embodiments, theCD8 antibody label and the CD127 antibody label are each a fluorescentlabel. In some further embodiments of any of the above, the sample isnot contacted with an antibody specific for the CD25 biomarker or forthe CD8 biomarker, or both. Preferably, the CD8+ and CD12?1^(◯1)− cellsare identified and isolated in a fluorescence activated cell sorter. Insome embodiments, of any of the above, the sample is a sample ofperipheral blood. Preferably, in some embodiments, at least 2, 4, 8, 10,or 20% of the CD8+CD12?1^(◯1)− T-cells, of the population are alsoCD2s+− or CD2s−. In other embodiments, the isolated population of Tregcells are substantially CD8+ and CD127−.

In other embodiments, the invention provides an expanded, isolatedpopulation of Treg cells which are substantially CD4+ and CD12?1^(◯1)−or CD8+ and CD12?1^(◯1)− wherein said expanded population is obtained bycontacting the isolated population as described above with alloantigenor anti-CD3/anti-CD28 antibodies in the presence of IL2 plus TGFbeta orrapamycin.

In other embodiments, the invention provides a pharmacologicalcomposition comprising isolated cell populations obtained as describedabove. In related embodiments, the invention provides for the use of apopulation of any one of the above claims in the manufacture of amedicament for suppressing the immune response in a subject in needthereof. The subject can have an immune disease or disorder, anautoimmune disease, a graft versus host reaction, an organ transplant,asthma, or allergy. In particular embodiments, the subject has Type Idiabetes.

Further in view of the above, the invention provides a method ofobtaining a population of anergic Treg cells, said method comprisingobtaining a sample containing Treg cells and sorting the cells accordingto the absence or presence of the CD127 biomarker, the CD4 biomarkerand/or the CD8 biomarker, and optionally the CD25 biomarker. In furthersuch methods the anergic Tregs are substantially FoxP3+ cells.

In yet other embodiments within this view, the invention provides amethod of obtaining an expanded population of anergic T-cells, saidmethod comprising the step of obtaining a sample containing Treg cells,expanding a CD4+CD12?1^(◯1) Treg or CD4⁸CD12?1^(◯1)⋅Treg cell populationtherein and sorting the cells according to the absence or presence orlevel of expression of the CD127 and CD4 or CD8 biomarkers wherein thesuppressive T″cell subset consists substantially of the expandedpopulation of CD8+cn12?1^(◯1)− Treg or CD4+CD12?1^(◯1)− T-cells. In somefurther embodiments, the cells of the expanded population are notfurther sorted with respect to the presence of absence of the CD25biomarker and the suppressive T″cell subset consists substantially ofCD4+CD12?1^(◯1)⋅T-cells, with at least 2% of the CD4+CD12?1^(◯1)−T-cells, being CD2s−. In other embodiments, if sorted according theirlevel of CD25 expression, CD25+ cells in addition to Cd25hi cells arepositively selected for.

In yet other embodiments within this view, the invention provides amethod of obtaining an expanded population of anergic T-cells, saidmethod comprising the step of obtaining an enriched T-cell sample andisolating and expanding or expanding and isolating a CD127Lo/− Treg cellpopulation therein. In some further embodiments, the cells of theexpanded population are not further sorted with respect to the presenceof absence of the CD25 biomarker and the suppressive T-cell subsetconsists substantially of CD4+CD12?1^(◯1)− T-cells, with at least 2% ofthe CD4+CD12?1^(◯1)− T-cells, being CD2S−. In other embodiments, ifsorted according their level of CD25 expression, CD25+ cells in additionto Cd25hi cells are positively selected for.

In another embodiment with this view, the invention provides a method ofobtaining an expanded population of anergic T-cells, said methodcomprising the step of obtaining a sample containing Treg cells, sortingthe cells according to the absence or presence of the CD127 and CD4 orCD8 biomarkers to obtain an isolated population of CD4+cn12?1^(◯1)− Tregcells and expanding the isolated population of CD4+CD12?1^(◯1)− Tregcells to obtain the expanded population of anergic T-cells. In yet otherembodiments of such, the cells of the expanded population are notfurther sorted with respect to the presence of absence of the CD25biomarker and the suppressive T″cell subset consists substantially ofCD4+CD12?1^(◯1)− T-cells, with at least 2% of theCD4+CD12?1^(◯1)⋅T-cells, being CD2s−.

Methods of Modulating an Immune Reaction Using Expanded Populations ofCI.H27h⋅/− Tregs,

In another aspect, the invention provides methods for producing anantigen-specific regulatory T cell enriched composition, and resultantcompositions and methods of use. In one embodiment, the inventionprovides a method of modulating an immune reaction in a subject, saidmethod comprising (a) obtaining a population of subject-compatible cellscontaining CD4+ and CD4⋅cells and CD127+ and CD127− cells and CD25+ andCD2S− cells; (b) producing an antigen-specific regulatory CDI2?1^(◯1) ⋅Tand/or FoxP3+ cell enriched composition from said population of cells byuse of methods described above and (c) introducing said composition intosaid subject to modulate said immune reaction in said subject. In somepreferred embodiments, the immune response is an autoimmune response andthe antigen is an autoantigen. In other embodiments, the immune responseis a graft vs. host immune response and the antigen is an autoantigen.In other embodiments, the immune response is allergy, asthma, tissue ororgan transplant rejection, or a graft vs. host immune response and theantigen is a purified or unpurified component of the allergentransplanted tissue or organ provoking the immune response. In preferredembodiments of any of the above, the enriched composition was notdirectly selected for on the basis of the presence or absence of CD25biomarker on the cells. In some embodiments, accordingly, the enrichedcomposition comprises at least 2%, 4%, 6%, 8% of CD2s− cells. In someembodiments, the enriched composition was also enriched for CD25+ cellsby sorting the cells on the basis of the presence or absence of the CD25biomarker on the cells.

In particular embodiments, the population of cells is obtained from saidsubject, obtained from a donor distinct from said subject, and/orharvested from peripheral blood. The population of cells obtainedcomprises antigen or autoantigen-specific regulatory T (Treg) cells, andmay be derived from any source in which antigen or autoantigen-specificTreg cells exist, such as peripheral blood, the thymus, lymph nodes,spleen, and bone marrow. In certain embodiments, the source of Tregcells may be from cadaveric tissue.

In other embodiments, the producing step comprises expanding saidantigen-specific regulatory T cells, and/or enriching theantigen-specific regulatory T cells from said obtained population ofcells.

In some embodiments, the expanding is achieved by contacting saidpopulation of cells with an antigen-specific regulatory T cellstimulatory composition.

In particular embodiments, the isolated CDI2?1^(◯1)−, CD4+cn12?1^(◯1)−and/or FoxP3+ T cells are isolated from said population of cells priorto said expanding step, or after said expanding step. In someembodiments, accordingly, the isolated cells comprise at least 2%, 4%,6%, 8% of CD2S− cells.

In particular embodiments, the stimulatory composition comprises an MHCclass II/autoantigenic peptide complex, a co-stimulatory agent or asecond regulatory T cell stimulatory agent. In other embodiments, theisolated CD127¹⁰¹−, CD4+CD127¹⁰¹-cells with or without the inclusion ofCD25 as an additional positive selection marker are isolated from saidpopulation of cells prior to said expanding step, or after saidexpanding step.

In yet another embodiment, the co-stimulatory agent is an agonistantibody (e.g., an agonist antibody which binds to CD28). In stillanother embodiment, the second stimulating agent is a cytokine, (e.g, aninterleukin, (e.g., interleukin-2)). In some embodiments, theco-stimulatory agent is an agonist antibody (e.g., an agonist antibodywhich binds to CD28 in the presence or absence of a second stimulatingagent which can be a cytokine (e.g., an interleukin, interleukin-2).

In particular embodiments, the stimulatory composition is immobilized ona substrate, such as a cell or bead.

The invention also provides compositions comprising a population ofcells wherein the cells are substantially antigen- or auto-antigenspecific regulatory CD12?1^(◯1)⋅, CD4+CD127¹⁰¹⋅T-cells and/or FoxP3+T-cells which comprise at least 2, 4, 6, 8, or 10% of CD2s− T cells.

The invention also provides compositions comprising a population ofcells wherein the cells are substantially antigen- or auto-antigenspecific regulatory CD12?1^(◯1)−, CD8+CD12?1^(◯1)⋅T-cells and/or FoxP3+T-cells which comprise at least 2, 4, 6, 8, or 10% of CD2s− T cells.

In particular embodiments, autoantigen-specific regulatory T cells arespecific for peptides presented in MHC class II molecules includingthose shown in Table A In other embodiments, the autoantigen-specificregulatory T cells are effective at modulating an autoimmune reactionwhen administered to a subject.

TABLE A MHC class II molecule/peptide(s) Autoimmune disease Autoantigenbound (SEQ ID NO:) Lupus erythematosus giantin golgin-245/p230golgin-160/GCP170 golgin-95/GM130 golgin-97 golgin-67 transferrin119-VVKKGTDFQLNQLEGKK (3) 119-VVKKGTDFQLNQLGKK (4)[see Freed et al., J. Immunol. (2000) 164: 4697-4705 A k (37-51 major;YVRFDSDVGEYRA VTE (5) (37-52 minor) Lvsozvme c (48-63)GDQSTDYGIFQINSRY (6) nucleoporin NUP155 RQVRFYSGVIEL (7) (120−)Saposin D (37−) LPDPYQKQCDDFVAE(8) 26S proteasome IFLDDPQAVSDVL (9)pl 12 (224−) 14-3-3 protein '8, KTAFDEAIAELD (10) (, 8, or T (95−)A k (146−) STQLIRNGDWTFQVLVMLEM (11) (I IO−) HHNTLVCSVTDFYPAKIKVR (12)Ig y I-chain (141−) SMVTLGCLVKGYFPEPVTVT (13) ThrombocytopeniaGPIIb/Illa HLA-DR purpura (Kuwana et al., J Clin Invest. 1998 Oct I;I02(7); 1393-402) platelet integrin Goodpasture's human glomerularsyndrome basement membrane Graves disease thyroglobulin thyroperoxi dasesodium-iodide symporter TSH receptor Type I diabetes Insulin, proinsulinDQ0601/insulin B ss5-15 mellitus aal-15: FVNQHLCGSHL VEAL (14)(see Ettinger and Kwok, J. Immunol. 1998 Mar I; 160(5):2365-73) HLA-DR3glutamic acid HLA-DR4 (DRBI*0401)/271-285 decarboxylase(PRLIAFTSEHSHFSL) (15), (GAD65) 116-130 (NILLQYVVKSFDRST) (16);HLA-DR4 (DRAI*0101)/356-370 (KYKIWMHVDAAWGGG) (17), 376-390 (KHKWKLNGVERANSV) (18), 481-495 (LYNIIKNREGYEMVF) (19),511-525 (PSLRVLEDNEERMSR) (20), 546-560 (SYQPLGDKVNFFRMV) (21),556-570 (FFRMVISNPAATHQD) (22), and 566-580 (ATHQDIDFLIEEIER) (23);HLA-DQ8/206-220 (TYEIAPVFVLLEYVT) (24) (see Peng, Y. Chin Med J2001; 114(10):229-242) tyrosine phosphatase IA-2 tyrosine phosphatase 2bIGRP Human protein: Q9UN79-SOX-13 protein (Type 1 diabetes autoantigenICA12) (Islet cell antigen 12). ICA69 Mysasthenia gravis Gravinmuscle nicotinic 121-126 (PAIFKSYCEIIVTHFP) (25), acetylcholine129-145 (EIIVTHFPFDEQNCSMK) receptor (AChR)(26) [seeJimmunol 159(3):1570-7] pl95- 212 (DTPYLDITYHFVMQRLPL)(27) [see Scand J Immun. 44(5):512-21] Pemphigus vulgaris desmoglein 1,desmoglein 3, Human desmocollin 1 (Dscl) bullous pemphigoid BP 180Autoimmune Formiminotransferase hepatitis cyclodeaminaseAutoimmine atrophic parietal cell H,K- corpus gastritis adenosinetriphosphatase (ATPase) Addison's diesease CYP21 CYP17 CYP11A1Rheumatoid arthritis endoplasmic reticulum molecular chaperoneimmunoglobulin binding protein (BiP) human cartilageHLA-DR4 (DRB1*0401)/aa259-271 glycoprotein-39 (PTFGRSFTLASSE) (28)(YKL40) (see Vos et al, Rheumatology (2000) 39:1326-133 I)type II collagen glucose-6-phosphate isomerase Multiple sclerosisalpha 13-Crystallin DRBI *1501 myelin HLA-DR4(DRB1 *0401)/97-108oligodendrocyte (TCFFRDHSYQEE) (29) glycoprotein (MOG)(see Forsthuber et al, J Immunol. 2001 Dec 15; 167(12):7119-25)Myelin basis protein 111-119 (SLSRFSWGA) (30) and 87-95 (MBP)(VVHFFKNIV) (31) presented in HLA- A2 and HLA-A24)[see JI, 172(8):5120-7] X2MBP Psoriasis Cytokeratin I7 cutaneouslymphocyte antigen (CLA) Autoimmune anion channel861-874 (CLAVLWVVKSTPAS) (32) hemolytic anemia protein band 3[see Blood 15;102(10):3800-6] Uveitis S-antigen341-354 (FLGELTSSEVATEV) (33) [see Int. Immun., 15(8):927-935]interphotoreceptor retinoid-binding protein (IRBP) HLA-B(B27PD)125-138 ALNEDLSSQTAADT (34) rsee Int. Immun., 15(8):927-9351

The invention also provides kits for producing a composition ofantigen-specific and antigen non-specific regulatory T cells. The kitcan include (a) CD127 antibody, (b) an antigen-specific or non-specificT cell receptor stimulatory agent; and (c) a costimulatory agent. Inparticular embodiments, the stimulatory agent is an MHC classII/autoantigenic peptide complex. In some embodiments, the costimulatoryagent is an agonist antibody, such as an antibody which binds to CD28.The kit may further comprise a second regulatory T cell stimulatingagent, such as a cytokine, such as an interleukin, such as interleukin-2or interleukin-IS. The stimulatory agent and said costimulatory agentcan be immobilized on a substrate, such as a cell or bead in someembodiments. The kit may also contain a CD4 antibody and/or a CD25antibody. The antibodies preferably are fluorescently or magneticallylabeled and monoclonal. The kit preferably will also containinstructions as to how to isolate CD127¹⁰¹⋅cells and how to store orformulate them for in vitro or in vivo use. Such kits may also provideinstructions as to how to select for the CDI2?1^(◯1)− cells using aCD127 antibody. In some embodiments, there is a proviso that the kitdoes not have means for detecting the CD25 marker.

The invention provides methods and compositions for ex vivo expansion oftherapeutic regulatory T cells, and resultant compositions and methodsof use. The expansion methods generally comprise the steps of: isolatingfrom a mixed population of T cells a subpopulation enriched inCD12?1^(◯1)T cells (Treg cells) by sorting the cells according to thepresence or absence or expression of biomarkers as set forth above;expanding the Treg cells of the subpopulation by contacting thesubpopulation with effective amounts of (i) a TCR/CD3 activator (ii) aTCR costimulator activator and (iii) IL-2, to obtain ex vivo expandedTreg cells, wherein the expanded Treg cells demonstrate immunesuppression, wherein the isolating step is typically prefaced byextracting the population from a person or patient, typically sufferingor in remission from an autoimmune or other immune (e.g., graft vs. hostdisease, asthma, allergy, transplant rejection) disease amenable totherapy as described herein. In some embodiments, the isolating from amixed population of T cells of a subpopulation enriched in CD4+CD12?1^(◯1)− T cells (Treg cells) by sorting the cells according to thepresence or absence or expression level of the CD127 biomarkers isperformed without regard to the presence or absence of CD25 or CD4 onthe cells. In other embodiments, the Treg cells are also enriched forCD25+ cells by selection according to the absence or presence of theCD25 biomarker.

In particular embodiments, the subpopulation comprises greater than 90,95, 98 or 99% CDI2?1^(◯1)⋅Treg cells; the isolation step step comprisesboth negative and positive immuno-selection and cell sorting; preferablythe expanding step effects at least a I00-fold expansion of thesubpopulation; the TCR/CD3 activator is a multivalent antibody or ligandfor TCR/CD3; the TCR costimulator activator is a multivalent antibody orligand for CD28, GITR, B7-1/2, CDS, ICOS, OX40 or CD40; the effectiveamount of IL-2 is 200 to 2500 IU IL-2/ml; and/or the Treg cells suppressproliferation of anti-CD3 or alloantigen stimulated CD2s− T cells invitro, or allergic immunity or autoimmunity, including graft-versus-hostdisease in vivo.

In more particular embodiments:

-   -   an effective amount of the ex vivo expanded Treg cells is        introduced into the patient diagnosed with diabetes mellitus and        presenting an indication of impaired glucose homoeostasis        selected from fasting plasma glucose (FPG), post-prandial        glucose (PPG), and glucose tolerance (GTT) and the introduction        provides a resultant improvement in the impaired glucose        homoeostasis, wherein the improvement is preferably selected        from an FPG of 110 mg/dL or less, a 2-hour PPG of 140 mg/dL or        less, and a GTT of 140 mg/dL or less 2 hours after a 75-g        glucose load;    -   the TCR/CD3 activator is an anti-CD3 antibody, and the TCR        costimulator activator is an anti-CD28 antibody, wherein the        anti-CD3 and anti-CD28 antibodies are immobilized on        paramagnetic beads provided in a Treg cell:bead ratio of between        1:1 and 1:2;    -   the TCR/CD3 activator and the expanded Treg cells are        antigen-specific, preferably wherein the TCR/CD3 activator is an        MHC-peptide multimer, wherein the peptide is a        diabetes-associated autoantigen peptide and the        diabetes-associated autoantigen is selected from glutamic acid        decarboxylase (GAD), an islet cell autoantigen (ICA) and        insulin, and the TCR costimulator activator is an anti-CD28        antibody.

The invention also provides methods and compositions for adoptivecellular immunotherapy comprising the step of introducing into a patientin need thereof an effective amount of the subject ex vivo expandedCD4+CD12?1^(◯1)− or CD4+FoxP3+ Treg cells or CD12?1^(◯1)− or FoxP3+ Tregcells. These methods generally comprise the steps of: extracting a mixedpopulation of T cells from a person; isolating from the population asubpopulation enriched in CD12?1^(◯1)− regulatory T-cells; expanding theregulatory T-cells of the subpopulation by contacting the subpopulationwith effective amounts of (i) a TCR/CD3 activator, (ii) a TCRcostimulator activator, and (iii) IL-2, to obtain ex vivo expandedregulatory T-cells; introducing into a patient an effective amount ofthe ex vivo expanded regulatory T-cells; and detecting a resultantsuppression of autoimmunity.

In particular embodiments, the person and patient is a patient diagnosedwith diabetes mellitus and presenting an indication of impaired glucosehomoeostasis selected from fasting plasma glucose (FPG), post-prandialglucose (PPG), and glucose tolerance (GTT); the subpopulationcomprises >98% Treg cells; the subpopulation comprises >98%CD4+CD12?1^(◯1)− Treg cells; the isolation step comprises negative andpositive immuno-selection and cell sorting; the expanding step effectsat least a 100-fold expansion of the subpopulation; the TCR/CD3activator is selected from a multivalent antibody or ligand for TCR/CD3;the TCR costimulator activator is a multivalent antibody or ligand forCD28, GITR, CDS, ICOS, OX40 or CD40L; the effective amount of IL-2 is200 to 2500 IUIL-2/ml; the Treg cells suppress proliferation of anti-CD3or alloantigen stimulated CD25.sup.− T cells, and/or the resultantsuppression of autoimmunity is detected as a resultant improvement inthe impaired glucose homoeostasis.

In more particular embodiments:

-   -   the improvement is selected from an FPG of 110 mg/dL or less, a        2-hour PPG of 140 mg/dL or less, and a GTT of 140 mg/dL or less        2 hours after a 75-g glucose load;    -   the TCR/CD3 activator is an anti-CD3 antibody, and the TCR        costimulator activator is an anti-CD28 antibody, wherein the        anti-CD3 and anti-CD28 antibodies are immobilized on        paramagnetic beads provided in a Treg cell:bead ratio of between        1:1 and 1:2; and/or the TCR/CD3 activator is an MHC-peptide        multimer, wherein the peptide is a diabetes-associated        autoantigen peptide and the diabetes-associated autoantigen is        selected from glutamic acid decarboxylase (GAD), an islet cell        autoantigen (ICA) and insulin, and the TCR costimulator        activator is an anti-CD28 antibody.

The population of cells may be obtained from the subject into which theisolated or isolated and expanded Treg-enriched composition issubsequently introduced. The subject can be any mammal in whichmodulation of an autoimmune reaction is desired. Mammals of interestinclude, but are not limited to: rodents, e.g. mice, rats; livestock,e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats; and primates, e.g.humans. In one embodiment, the subject is an animal model of anautoimmune disease. There are numerous, established animal models forusing T cell epitopes of autoantigens to induce tolerance, includingmultiple sclerosis (EAE: experimental autoimmune encephalomyelitis),myasthenia gravis (EMG: experimental myasthenia gravis) and neuritis(EAN: experimental autoimmune neuritis). In another embodiment, thesubject is a human afflicted with an autoimmune disease or disorder,such as any of the diseases/disorders listed in Table A

In an alternate embodiment, the population of cells is obtained from adonor distinct from the subject. The donor is preferably syngeneic, butcan also be allogeneic, or even xenogeneic provided the cells obtainedare subject-compatible in that they can be introduced into the subject,optionally in conjunction with an immunosuppressive therapy, withoutresulting in extensive chronic graft versus host disease (GvHD).Allogeneic donor cells are preferably human-leukocyte-antigen(HLA)-compatible, and are typically administered in conjunction withimmunosuppressive therapy. To be rendered subject-compatible, xenogeniccells may be subject to gamma irradiation or PENl 0 treatment (Fast, L Det al, Transfusion. 2004 February; 44(2):282-5).

The producing step can provide a predetermined antigen- orautoantigen-specific regulatory T cell enriched composition from saidpopulation of cells, preferably specific for a predetermined antigen orautoantigen associated with the targeted allergic or autoimmunereaction, preferably predetermined to be associated with the targetedallergic or autoimmune reaction. In particular embodiments, theproducing step comprises expanding said antigen-specific regulatory Tcells, and/or enriching said autoantigen-specific regulatory T cellsfrom said obtained population of cells.

An antigen or autoantigen-specific regulatory T (Treg) cell enrichedcomposition is one in which the percentage of antigen orautoantigen-specific CD12?1^(◯1)− Treg cells is higher than thepercentage of antigen or autoantigen-specific CD12?1^(◯1)− Treg cells inthe originally obtained population of cells. In particular embodiments,at least 75%, 85%, 90%, 95%, or 98% of said cells of the composition areantigen or autoantigen-specific regulatory T cells. In particularembodiments, the producing step comprises expanding the antigen-specificregulatory CD12?1^(◯1)− T cells, and/or enriching saidautoantigen-specific regulatory CD12?1^(◯1)− T cells from said obtainedpopulation of cells. In some embodiments, the composition is furtherenriched by positive selection for CD4 and/or CD25 biomarkers.

In particular embodiments, the regulatory CD12?1^(◯1)− T cells areenriched from said population of cells prior to said expanding step, orafter said expanding step. CD12?1− Treg cells can be enriched bytargeting for selection of cell surface markers specific for immunesuppressive CD12?1^(◯1)− Tregs and separating using automated cellsorting such as fluorescence-activated cell sorting (FACS), solid-phasemagnetic beads, etc, as is known to one of ordinary skill in the art(see, U.S. Patent Application Publication No. 2005/0186207) which isassigned to the same assignee as the present invention and incorporatedby reference in its entirety. To enhance enrichment, positive selectionfor T-reg cells may be combined with selection against cells comprisingsurface makers specific to non-Treg cell types, such as depletion ofCD8, CDI lb, CD16, CD19, CD36 and CD56-bearing cells as known to one orof ordinary skill in the art.

In particular embodiments, the expanding is achieved by contacting thepopulation of cells with an antigen- or autoantigen-specific regulatoryCDI2?1^(◯1)− T cell stimulatory composition. The antigen orautoantigen-specific regulatory CDI2?1^(◯1)− T cells are preferablyexpanded at least 50-fold, and preferably at least 100, 200, 300, 500and 800-fold. Antigen and autoantigen-specific regulatory CDI2?1^(◯1)− Tcell stimulatory compositions promote the survival, growth, and/orexpansion of the antigen or autoantigen-specific regulatory CDI2?1^(◯1)−T cells that express T cell receptor(s) that recognize the desiredantigen or autoantigen.

Preferred stimulatory compositions stimulate the CDI2?1^(◯1)− T cell byantigen-specifically binding and activating the T cell receptor complex.A variety of antigen-specific TCR-binding reagents may be used,including cross-linked peptide-bound MHC molecules, antibodies, andmimetics. In a preferred embodiment, the compositions comprises an MHCclass I/autoantigenic peptide complex, particularly an aggregate of suchMHC/peptide complexes. These complexes comprises at least theextracellular peptide binding domain of an MHC class II molecule inwhich is functionally bound an autoantigenic peptide. The complexes canbe in solution or suspension or immobilized on a substrate, such aspresented on the surface of a cell, particularly an APC. Numerousapplicable methods are known in the art for generating functional MHCclass I/peptide complexes, such as may be found in literu In oneembodiment, the autoantigenic peptide is a peptide of the naturallyoccurring autoantigen that is capable of complexing with an MHC class IImolecule. Exemplary MHC class II molecules/peptide complexes are listedin Table A In an alternative embodiment, the autoantigenic peptide is amimotope peptide capable of complexing with an MHC class II molecule.

In another embodiment the autoantigenic peptide is a mimotope peptidethat is capable of complexing with an MHC class II molecule. Mimotopepeptides are described in the literature, further below, and inExamples 1. Protocols for using autoantigen peptides to expand Tregsfrom otherwise conventional T cells include the use ofautoantigen-specific MHC-peptide tetramers, peptide-pulsed DCs(Yamazaki, et al, 2003, J Exp Med 198:235-47) or artificial APCs (Mauset al. Nat. Biotechnol. 20:143-8, 2002) to expand Tregs from patientsindependent of the cell surface phenotype. In addition, a combination ofin vitro and in vivo approaches can enhance the effects of the therapy.For example, recent studies have shown that administration of selfantigens, altered peptide ligands and even non-specific stimuli such asFcR non-binding anti-CD3 mAbs can promote antigen-specific Treg activity(Apostolou et al. J. Exp. Med. 199:1401-8, 2003; Belghith et al. Nat.Med. 9:1202-8, 2003). Hence, combining in vivo immunization to inducethe Tregs with ex vivo expansion or visa versa may be advantageous.

The stimulatory composition may further include one or more additionalagents, e.g., a costimulatory agent, a second regulatory T cellstimulatory agent, or agents that generally promote the survival and/orgrowth of T cells.

The costimulatory agent is an antibody or ligand specific for a TCRcostimulator, such as CD28 or GITR, as described below. In particularembodiments, the costimulatory agent is an agonist antibody, such as anagonist antibody which binds to CD28.

The stimulatory composition alternatively comprises a second regulatoryT cell stimulatory agent. Exemplary stimulatory agents includegranulocyte colony stimulating factor, interleukins such as IL-2, IL-6,IL-7, IL-13, and IL-15, and hepatocyte growth factor (HGF). Inparticular embodiments, the second stimulating agent is a cytokine, suchas an interleukin, such as interleukin-2.

In some embodiments, one or more components of the stimulatorycomposition is immobilized on a substrate, such as a cell or bead. Cellssuitable for use as substrates include artificial antigen-presentingcells (aAPCs) (Kim, J V et al, Nat Biotechnol. 2004 April; 22(4):403-10;and Thomas, A K et al, Clin Immunol. 2002 December; 105(3):259-72).Beads can be plastic, glass, or any other suitable material, typicallyin the 1-20 micron range. Paramagnetic beads are preferred.

Optimal concentrations of each component of the stimulatorycompositions, culture conditions and duration can be determinedempirically using routine experimentation.

The expanded and/or enriched antigen- or autoantigen-specific regulatoryCD12?1^(◯1)− T cells are introduced into the subject to modulate animmune or autoimmune reaction. For example, the subject may be afflictedwith a disease or disorder characterized by having an ongoing orrecurring autoimmune reaction, such as the diseases/disorders listed inTable A In particular embodiments, the said modulating comprisesinhibiting. CD12?1^(◯1)− Tregs may serve as a “Trojan Horse” to deliversuppressive or other biologic factors to sites of inflammation, such asIL-4 (Yamamoto et al. J. Immunol. 166:4973-80, 2001), stem cell growthfactors, angiogenesis regulators, genetic deficiencies, etc. Forexample, overexpression of foxp3 has been shown to transform otherwisepathogenic T cells into Tregs (Jaeckel et al. Diabetes. 2004 Dec. 10;[Epub]), and polyclonally expanded Tregs can be transduced with genesencoding an antigen-specific TCR plus FoxP3 to generate potentantigen-specific Tregs in very high numbers and efficiency (Mekala, etal., Blood. 2004 Nov. 4; [Epub]). Thus, these antigen-specificapproaches decrease the requirement for high cell numbers whilemaximizing Treg specificity and function.

Antigen-specific CD12?1^(◯1)− Tregs are particularly indicated ininfectious diseases in which the pathogenicity of the infections is nota result of the cytopathic effects of the pathogen but rather the tissuedamage caused by the immunoinflammatory response to the infectiousagent. In diseases, such as hepatitis C or HSY-induced cornealinflammation, CD4+CD12?1^(◯1)− Treg therapy provides a uniqueopportunity to control viral-induced immunoinflammatory disease (Suvaset al. J. Immunol. 172: 4123-4132, 2004). Viruses, such as Coxsackie,are known to cause pancreatitis and have been associated with thedevelopment of Type 1 Diabetes. Thus, CD12?1^(◯1)− Tregs that targetexpressed viral antigens can be used to suppress local tissue damagecaused by the infection and reduce the inflammation that incitesautoimmune disease development.

The invention also provides compositions comprising a population ofcells wherein at least 50% of said cells of said composition are natural(nontransformed), preferably expanded antigen or autoantigen-specificCD12?1^(◯1)− Treg cells, wherein the antigen or autoantigen-specificityis preferably predetermined, preferably predetermined to a targetedimmune or autoimmune reaction antigen. The compositions are made by themethods described herein. The percentage of the antigen orautoantigen-specific regulatory CD12?1^(◯1)− Treg cells in thecomposition can be ascertained using the methodology described in theExamples. Preferably, at least 75%, 85%, 90%, 95%, or 98% of said cellsof the composition are antigen or autoantigen-specific regulatory Tcells.

In addition, the autoantigen-specific regulatory CD12?1^(◯1)− T cells,in some embodiments, are specific for an MHC class II molecule/peptidecomplex listed in Table A.

In some embodiments, the autoantigen-specific regulatory CD12?1^(◯1)− Tcells are effective at modulating an autoimmune reaction whenadministered to a subject. Effective and optimized dosages and treatmentregimes using the expanded and/or enriched autoantigen-specificregulatory cells are informed from vast clinical experience withexisting T-cell infusion therapies, and can be further determinedempirically.

The subject methods find use in the treatment of a variety of differentconditions in which the modulation of an aberrant immune response in thehost is desired. By aberrant immune response in a host is meant anyimmune reaction in a subject characterized as an unwanted immune orautoimmune response (e.g., an autoimmune disease). In general,autoimmune responses occur when the immune system of a subjectrecognizes self-antigens as foreign, leading to the production ofself-reactive effector immune cells. Self reactive effector immune cellsinclude cells from a variety of lineages, including, but not limited to,cytotoxic T cells, helper T cells, and B cells. While the precisemechanisms differ, the presence of autoreactive effector immune cells ina host suffering from an autoimmune disease leads to the destruction oftissues and cells of the host, resulting in pathologic symptoms.Numerous assays for determining the presence of such cells in a host,and therefore the presence of an autoimmune disease, such as an antigenspecific autoimmune disease in a host, are known to those of skill inthe art and readily employed in the subject methods. Assays of interestinclude, but are not limited to, those described in: Autoimmunity. 2003September-November; 36(6-7):361-6; J Pediatr Hematol Oncol. 2003December; 25 Suppl 1:S57-61; Proteomics. 2003 November; 3(11):2077-84;Autoimmun Rev. 2003 January; 2(1):43-9.

By treatment is meant that at least an amelioration of the symptomsassociated with the aberrant immune response in the host is achieved,where amelioration is used in a broad sense to refer to at least areduction in the magnitude of a parameter, e.g. symptom, associated withthe condition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g. prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.

A therapeutically effect amount of a composition or cell population isan amount which is sufficient to treat or ameliorate the subjectcondition.

A variety of hosts are treatable according to the subject methods. Incertain embodiments, such hosts are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys). In preferred embodiments, the hostswill be humans.

In further embodiments, the methods include a step of diagnosing thepresence of an autoimmune disease to be treated. By diagnosing is meantthat the autoimmune response of a subject is generally classified, e.g,diabetes mellitus, SLE, MS, etc. Further, at least one autoantigen maybe identified to which the aberrant immune response is directed.

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. In certain embodiments, the kits include at least aCD127 antibody, and an antigen specific regulatory T cell stimulatorycomposition. In other embodiments, the kit includes another regulatory Tcell stimulating agent, such as a cytokine, such as an interleukin, suchas interleukin-2 or interleukin-15. In certain embodiments, the kits mayfurther include reagents for performing the antigen specific regulatoryT cell expansion step, including culture dishes or flasks, culturemedium, or any necessary buffers, factors, etc. In yet otherembodiments, the kits include the means to harvest the sample containingthe regulatory T cells and the reagents necessary to perform regulatoryT cell enrichment/purification. The antibody may be labeled or the kitmay provide reagents for labeling the antibody. In some embodiments ofthe above, the kits further comprise a CD4 and/or a CD25 antibody and,optionally, selecting for cells which are CDI2?1^(◯1)−, CD4+.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

In some embodiments, the stimulatory agent is an MHC class1/autoantigenic peptide complex. Exemplary MHC class IImolecules/peptide complexes are listed in Table A In other embodiments,the stimulatory agent is an antigen which prompts an unwanted immuneresponse in the subject or patient.

The costimulatory agent can be an antibody or ligand specific for a TCRcostimulator, such as CD28 or GITR, as described below. In particularembodiments, the costimulatory agent is an agonist antibody, such as anantibody which binds to CD28. In some embodiments, the stimulatory agentand said costimulatory agent are immobilized on a substrate, such as acell or bead.

The invention also provides methods and compositions for ex vivoexpansion of therapeutic regulatory CD12?1^(◯1)⋅T cells, and the use ofsuch expanded Treg cells for adoptive cellular immunotherapy to suppressautoimmunity.

The expansion methods generally comprise first extracting a mixedpopulation of T cells from a person or patient, and isolating from thepopulation a subpopulation enriched in CD12?1− Treg cells. To maximizeefficacy, the subpopulation is enriched to at least 90%, preferably atleast 95%, and more preferably at least 98% CD12?1^(◯1)− Treg cells.Cells are generally enriched by targeting for selection cell surfacemarkers specific for immune suppressive CD12?^(◯1)− Tregs and separatingusing automated cell sorting such as fluorescence-activated cell sorting(FACS), solid-phase magnetic beads, etc. To enhance enrichment, positiveselection may be combined with negative selection against cellscomprising surface makers specific to non-Treg cell types, such as bydepletion of CD8, CD11b, CD16, CD19, CD36 and CD56-bearing cells, and asexemplified below.

The CD12?1^(◯1)− Treg-enriched subpopulation is then expanded ex vivo byculturing the cells in the presence of effective amounts of a TCR/CD3activator, a TCR costimulator activator, and IL-2. The TCR/CD3 activatoris selected from a multivalent antibody or ligand for TCR/CD3, includingantigen non-specific activators such as an anti-CD3 antibody, andantigen-specific activators, such as an MHC-peptide multimer (see, e.g.Yee, et al., Adoptive T cell therapy using antigen-specific CD8+ T cellclones for the treatment of patients with metastatic melanoma: In vivopersistence, migration, and antitumor effect of transferred T cells.Proc Natl Acad Sci USA, Dec. 10, 2002; 99(25): 16168-16173; Butterfield,et al., T-Cell responses to HLA-A*0201 immunodominant peptides derivedfrom a-fetoprotein in patients with hepatocellular cancer, Clin. CancerRes., Dec. 1, 2003; 9(16): 5902-5908; and Yee, et al., Isolation of highavidity melanoma-reactive CTL from heterogeneous populations usingpeptide-MHC tetramers, J Immunol, 1999, 162: 2227-223), wherein thepeptide is typically an autoimmune disease associated peptide, such as adiabetes-associated autoantigen peptide wherein suitablediabetes-associated autoantigens include glutamic acid decarboxylase(GAD), an islet cell autoantigen (ICA) and insulin, wherein combinationsof such peptides may also be used.

The costimulator activator can be a multivalent antibody or ligandspecific for a TCR costimulator, preferably CD28 or GITR (Shimizu etal., Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaksimmunological self-tolerance, Nat Immunol. 2002 February; 3(2): 135-42.Epub 2002 Jan. 22; Tone et al., Mouse glucocorticoid-induced tumornecrosis factor receptor ligand is costimulatory for T cells, Proc NatlAcad Sci USA 2003 Dec. 9; 100(25):15059-64. Epub 2003 Nov. 7), thoughalternative TCR costimulators such as CDS, ICOS, OX40 and CD40L may alsobe targeted where suitable expansion is so obtained, as may bedetermined empirically. To promote activation and expansion, the TCR/CD3and TCR costimulator activators can be typically immobilized on a3-dimensional solid surface, such as a host cell (e.g. Thomas et al,December 2002, Clin Immunol 105, 259-72) or bead. In a particularembodiment, the activators may be immobilized on paramagnetic beadsprovided in a Treg cell:bead ratio of between 2:1 and 1:5, preferablybetween 1:1 and 1:3. Optimal bead size can be empirically determined,though typically the size falls in the range of 1 to 20 microndiameters.

The IL-2 is typically presented in recombinant form, wherein effectiveamounts of IL-2 can be typically 200 to 2500 IU IL-2/ml. Maximalexpansions are determined empirically and will vary by cell type,incubation conditions, etc. For embodiments, maximal expansions may beabout 300, 500 and 800-fold.

The suppressive function of the expanded CD12?1^(◯1)− Treg cells may bedetected in vitro or in vivo. For example, in vitro, the expandedCD12?1^(◯1)− Treg cells may be shown to suppress proliferation of CD2S−T cells stimulated with anti-CD3 in the presence of Fe-receptor-bearingcells, or CD2S− T cells stimulated with irradiated allogeneicsplenocytes. Suitable exemplary in vivo animal model and human clinicalimmune suppression protocols are described in U.S. Patent ApplicationPublication No. 2005/0186207 which is herein incorporated by referencewith respect to same.

In some embodiments, the TCR/CD3 activator and the expanded CD12?1^(◯1)−Treg cells are autoantigen-specific. For example, in a particular suchembodiment, an effective amount of the ex vivo expanded CD12?1^(◯1)−Treg cells introduced into the patient diagnosed with diabetes mellitus(see, e.g. Mayfield et al., Diagnosis and classification of diabetesmellitus: new criteria, Am Fam Physician. 1998 Oct. 15; 58(6):1355-62,1369-70) and presenting an indication of impaired glucose homoeostasis,such as fasting plasma glucose (FPG), post-prandial glucose (PPG), andglucose tolerance (GTT) provide a resultant improvement in the impairedglucose homoeostasis, particularly wherein the improvement is selectedfrom an FPG of 110 mg/dL or less, a 2-hour PPG of 140 mg/dL or less, anda GTT of 140 mg/dL or less 2 hours after a 75-g glucose load.

Accordingly, the invention provides methods and compositions foradoptive cellular immunotherapy comprising introducing into a patient inneed thereof an effective amount of the subject ex vivo expandedCD12?1^(◯1)− Treg cells. These applications generally involvereintroducing expanded CD12?1^(◯1)− Treg cells extracted from the samepatient, though the methods are also applicable to adoptive cellularimmunotherapy for treatment of graft-versus-host disease associated withtransplantation, particularly bone marrow transplantation usingCD12?1^(◯1)− Tregs derived from donor tissue.

Adoptive transfer of CD12?1^(◯1)− Tregs expanded as disclosed herein canbe effective to suppress a wide variety of pathogenic autoimmuneresponses, including diabetes, GVHD, Lupus, rheumatoid arthritis,psoriasis, multiple sclerosis, degenerative heart disease (e.g. ZiadMallat, et al. Induction of a Regulatory T Cell Type 1 Response Reducesthe Development of Atherosclerosis in Apolipoprotein EBKnockout Mice,Circulation. 2003 Sep. 9; 108(10):1232-7), inflammatory bowel disease(Crohn's disease), etc.

In adoptive cell transfer protocols, a mixed population of T cells isinitially extracted from a target donor. Depending on the application,the T cells may be extracted during a period of remission, or duringactive disease. Typically this is done by withdrawing whole blood andharvesting granulocytes by leukapheresis (leukopheresis). For example,large volume leukapherisis (LVL) has been shown to maximize bloodleukocyte yield. Harvests reach 20×10⁶ cells/L using a continuous flowapheresis device (Spectra, COBE BCT). Symptoms of hypocalcemia areavoided by a continuous infusion of calcium administrated throughoutleukapheresis. Typically 15-45 liters of fluid corresponding to about 4total blood volumes are harvested during a period of time ranging fromabout 100 to 300 minutes.

The harvested lymphocytes are separated by flow cytometry or other cellseparation techniques based on Treg-specific cell markers such as CD127and expanded as described herein, and then transfused to a patient,typically the cell donor (except in GVHD where the donor and recipientare different), for adoptive immune suppression. Alternatively, thecells may be frozen for storage and/or transport prior to and/orsubsequent to expansion. In some embodiments for antigen non-specificexpansions, approximately 10⁹ to 10¹¹ Tregs are transfused; forantigen-specific expansions, therapeutically effective transfusionstypically may use about 10′ to 10⁹ Treg cells.

IHetlmd of Sorting ceHs.

The biomarkers used for positive or negative selection of the regulatoryT cells of the present invention may be identified by immunoselectiontechniques known to those in the art which utilize antibodies including,but not limited to, fluorescence activated cell sorting (FACS), magneticcell sorting, panning, and chromatography. Immunoselection of two ormore markers on activated T cells may be performed in one or more steps,wherein each step positively or negatively selects for one or moremarkers. When immunoselection of two or more markers is performed in onestep using FACS, the two or more different antibodies may be labeledwith different fluorophores.

Methods of sorting cells are well known to persons of ordinary skill inthe art. Cell sorters generally are capable of separating a complexmixture of cells into fractions of a single cell type. Typically, thecells to be sorted are introduced as a thin jet of carrier liquidemanating from a small nozzle orifice. Shortly after leaving the nozzle,the fluid passes through the waist of one or more tightly focused laserbeams. The scattered and fluorescence light from these interactions canbe collected and analyzed to determine if there are events (e.g., thepresence of a fluorescence signal indicating that a fluorophore-labeledmonoclonal antibody is bound to the surface of a cell) that prompt thesorting of the cell by various means. More than one label can bemonitored at a time. FACS (fluorescence activated cell sorters) caneasily analyze cells at speeds greater than 200,000 events per second.Generally, the physics of the carrier fluid, however, and the statisticsof distributing the cells among the droplets limits sort rates to about50,000 cells per second. This combination of speed and reliableseparation allows individual cells to be isolated for other uses.

Magnetic cell sorting may be performed using super-paramagneticmicrobeads composed of iron oxide and a polysaccharide coat. Preferablythe microbeads may be approximately 50 nanometers in diameter, and havea volume about one-millionth that of a typical mammalian cell. Themicrobeads are preferably small enough to remain in colloidalsuspension, which permits rapid, efficient binding to cell surfaceantigens. The microbeads preferably do not interfere with flowcytometry, are biodegradable, and have negligible effects on cellularfunctions. The antibody coupling to the microbeads may be direct orindirect, via a second antibody to a ligand such as fluorescein.

Methods of Administration of the Isolated and Isolated, Expanded CellPopulations.

The cells can be administered in a variety of ways. By way ofnonlimiting example, the cells may be delivered intravenously, or into abody cavity adjacent to the location of an immune response to besuppressed, such as the intraperitoneal cavity, or injected directlywithin or adjacent to the site of the immune reaction. Intravenousadministration, for example, is advantageous in the treatment of manysuch conditions.

The medicaments and pharmaceutical compositions may be formulated usingconventional pharmaceutically acceptable parenteral vehicles foradministration by injection. These vehicles may be nontoxic andtherapeutic, and a number of formulations are set forth in Remington'sPharmaceutical Sciences. Nonlimiting examples of excipients are saline,Ringer's solution, saline-dextrose solution, and Hank's balanced saltsolution. Pharmaceutical compositions may also contain minor amounts ofadditives such as substances that maintain isotonicity, physiologicalpH, and stability.

The medicaments and compositions may be in unit dose format. Generally,the unit dose will contain a therapeutically effective amount ofCD12i⁰¹-regulatory T-cells. The amount will generally depend on the age,size, gender of the patient, the condition to be treated and itsseverity, the condition of the cells, and their original characteristicsas obtained from the donor of the sample. Methods of titrating dosagesto identify those which are therapeutically effective are known topersons of ordinary skill in the art. Generally, a therapeuticallyeffective amount of the cells can be from 10⁷ to 10¹¹.

The following examples are included to illustrate the invention andmethods used in practicing the invention, and not to limit theinvention.

EXAMPLES Example 1. Antibodies

Human Antibodies: PE-conjugated anti-CD127, APC-conjugated anti-CD25,PerCP-conjugated anti-CD4 used for staining and in sorting was providedby Becton-Dickenson (BD Pharmingen, San Diego, Calif.).Alexa488-conjugated anti-FoxP3 was purchased from BioLegend (San Diego,Calif.) and intracellular staining was performed according tomanufacturer's instructions as modified as follows: 5×10⁵ cells werestained with cell surface markers for 30 min at 4° C. and fixed for 30min using Ix Fix/Perm buffer. After 3 washes cells were permeabilized inPerm buffer with DNase I (Sigma-Aldrich, St. Louis, Mo.) for 30 minfollowed by 3 washes. Then cells were blocked with human IgG and stainedwith anti-human FoxP3-Alexa488 conjugated (BioLegend, San Diego, Calif.clone 206D). The following anti-mouse antibodies were purchased from theindicated sources: anti-CD4, anti-CD25 and mouse IgG1 (isotype control)(BD Pharmingen, San Diego, Calif.); and anti-IL-7R (eBioscience, SanDiego, Calif.).

Example 2. Subjects

A total of 16 patients with longstanding type I diabetes mellitus werestudied. Patients (age range 16-56 years, mean age 34, with a diseaseduration longer than 5 years) were recruited from the Barbara DavisCenter for Childhood Diabetes, Denver, Colo., USA Diagnosis of type Idiabetes was made primarily by the presence of biochemicalautoantibodies or presentation of hyperglycemia with ketosis inchildhood. None of the diabetic subjects had severe nephropathy orneuropathy. As controls, 10 subjects (age range 20-50 years, mean age29) with no family history of diabetes mellitus were also tested. Bloodsamples were obtained with informed consent under Institutional ReviewBoard approved protocols at either the University of Colorado HealthSciences Center or UCSF as needed.

Example 3. Sorting of CD4+ T Cell Subsets for Flow Cytometry andFunctional Studies

Human T cells were isolated from leukopacs (Blood Centers of thePacific). In some cases, negative selection using RosetteSep human CD3depletion Cocktail (Stemcell Technologies, Seattle, Wash.) wasperformed. 100-120×10⁶ PBMC cells were washed once, counted andresuspended in sorting buffer (PBS+ 0.1% BSA+ 1 mM EDTA) at 100×10⁶ perml in a 15 ml conical tube. After addition of 10/1 million cells volumePerCP conjugated anti-CD4, 1μ1/1 million cells PE conjugated anti-CD127,and 0.7μ1/million cells APC conjugated anti-CD25 antibodies, the cellsuspension was mixed gently and incubated at 4 C for 30 min. Cold FACSsorting buffer was added up to a volume of 15 ml, T cells were pelletedand resuspended at 20×10⁶ per ml. Labeled T cells were sorted using anAria high-speed cell sorter. The sort gates for the various T cellsubsets were set to include only those events exhibiting theCD4-specific fluorescence that were also within the lowest densityregion of a scatter plot. This amounted to between 11.4% and 33.9% (mean20.87%) (n=22) of the total number of events for the CD4+ T cells. Basedon the CD4 gate, cells were then further gated based on CD127 and/orCD25 expression (CD4+CD127+¹−cn25+− and CD4+CD127+− alone independent ofCD25 as well as CD25hi conventional Tregs). Cells were collected into100% human AB serum (Cambrex, Walkersville, Md.) and washed once withmedia (RPMI/5% human serum) until ready to be plated in suppressionassay. Sorted Treg were 95-98% CD4+CD25hi with a typical yield of5-12×10⁵ T cells per sort whereas CD4+CD127-CD25+ cells had a typicalyield of 0.9-1.2×10⁶ and 98% purity.

Example 4. Isolation of CD4+CD25hi and CD4+cn25neg Cells for GeneChipArrays

Human CD4+ T cells were isolated by negative selection from leukopacs(Stanford University Blood Center) using RosetteSep Human CD4+ T cellCocktail (Stemcell Technologies, Seattle, Wash.). 7.5-1.25×10⁸ CD4+ Tcells (>90% purity by FACS) were washed once, counted and resuspended inFACS staining buffer (PBS+ 0.1% BSA) at 10×10⁶ per ml in a 50 ml conicaltube. After addition of 1/90 volume Cy-5 conjugated anti-CD4 (BDPharmingen) and 1/100 FITC-conjugated anti-CD25 (DakoCytomation,Chicago, Ill.) antibodies, the cell suspension was mixed gently andincubated on ice for 45 min. Cold FACS staining buffer was added to avolume of 50 ml, T cells were pelleted and resuspended at 20×10⁶ per ml.Labeled T cells were incubated on ice for 45 min and then submitted toflow sorting on a DakoCytomation MoFlo high-speed cell sorter. The sortgates were set to include only those events exhibiting the highestlevels of CD25-specific fluorescence (CD4+CD25hi cells) or lowest levels(CD4+CD25neg cells) that were also within the lowest density region of ascatter plot. This amounted to between 0.8% and 1.4% of the total numberof events for the CD4+ T cells for each subset.

Example 5. RNA Isolation

Total RNA was isolated from Treg using the total RNA isolation protocolfrom the RNA RT-PCR Miniprep kit (Stratagene, La Jolla, Calif.) with thefollowing modifications: 100,000 cells were lysed in 150 μl of lysisbuffer. To digest DNA, two units of DNAse were added/ug nucleic acid,and Phenol/CHCb (Sigma-Aldrich, St. Louis, Mo.) was used to purify totalRNA followed by ethanol precipitation. The quantity of total RNA wasmeasured using Nanodrop ND 100 (Nanodrop Technologies, Rockland, Del.).100 ng of each RNA sample was used for target labeling by a two-roundamplification protocol. This protocol was modified from the Affymetrixeukaryotic small sample prep by using 6 pMol of T7 primer and 3 μg/μ1 ofthe random primer.

Example 6. GeneChip Arrays and Data Analysis

A total 16 of human HG-U133A GeneChip arrays were used in this study(Affymetrix, Santa Clara, Calif.). Ten ug of fragmented cRNA perGeneChip hybridization were processed on the Affymetrix Fluidic station450 (Affymetrix, Santa Clara, Calif.) and GeneChip scanner GCS2500(Hewlett-Packard Company, Palo Alto, Calif.). Gene expression profilewas analyzed with MAS5.0 (Microarray Suite version 5.0 (Affymetrix,Santa Clara, Calif.) was used for data acquisition and normalization.Present genes were defined by selecting genes that were present in 3 of4 arrays. Signal intensities of all present genes for the activated andcontrol groups were combined and analyzed by T-test. Significant geneswere selected with p<0.05. The “signal log ratio” (SLR) and “Increase”or “Decrease” call was generated by comparison analysis MAS5.0 and thenused for calculating fold changes between groups. We selected 9 out of16 pair-wise comparisons for particular genes that showed increase ordecrease at fold change>2.0. In the second step, signal intensities ofpresent genes were analyzed with Significance Analysis of Microarrays(SAM) was applied for analyzing and determining the gene list basednumber of significant genes that were identified by T-test and foldchange. The final significant genes were combined from the above twosteps. Two-dimensional hierarchical clusters are generated usingGeneSpring 6.0 software (Silicon Genetics, Redwood City, Calif.).

Example 7. Real-Time PCR Analysis

RNA was isolated using RNeasy mini kits (Qiagen, Valencia, Calif.)according to the manufacturer's instructions. For cDNA synthesis, 500 ngtotal RNA was transcribed with cDNA transcription reagents usingSuperScriptlll reverse transcriptase and oligo(dT)12-18 (InVitrogen,Inc.), according to the manufacturer's instructions. Gene expression wasmeasured in real-time with the GeneAmp 7900 Sequence Detection System(Applied Biosystems) using primers and QuantiTect SYBR Green PCR Kitpurchased from Qiagen. The expression level of a gene in a given samplewas represented as 2−−⋅\Act whereti.ACT=[ACTcexperimental)]−[ACTcmedium)] and ACT=[CTcexperimental)]−[CTchousekeeping)]. Data is presented normalizedto the glyceraldehyde phosphate dehydrogenase (GADP).

Example 8. Suppression Assays

Suppression assays were performed in round bottom 96-well microtiterplates. 100,000 responder PBMC from same cell source as sortedpopulations, 30,000 sorted cells (one of 7 different sorted subtypesbased on CD25 and/or CD127 expression), 100,000 allogeneic irradiatedCD3-depleted PBMC were added as indicated. Responder ratio indicatedrefers to Treg to responder where 1 sorted: 1 responder is 30,000 sortedcells: 100,000 PBMC responder cells. APC consisted of allogeneic PBMCdepleted of T cells using StemSep human CD3+ T cell depletion permanufacturer's recommendations (StemCell Technologies, Seattle, Wash.)and irradiated with 40 Gy. Cells were plated in the following order in50 ul per well: sorted cells, responders, APC. No additional stimuluswas added to the wells, however, additional media was added to each wellso the final volume was 200 ul per well. Wells surrounding culture wellswere filled with PBS in order to prevent evaporation. T cells wereincubated for 7 days at 37° C. in 5% CO2. Sixteen hours before the endof the incubation, 1 uCi ³H-thymidine was added to each well. Plateswere harvested using a Tomtec cell harvester and ³H-thymidineincorporation was determined using a 1450 microbeta Wallac Trilux liquidscintillation counter.

Example 9. Antibody Staining and FACS Analysis

Five×10⁴ T cells per sample were washed once with FACS staining buffer(PBS+0.1% BSA) and resuspended in I 00 μL buffer. I μL offluorescence-conjugated specific antibodies (1 μg/million T cells) wasadded, T cells were lightly vortexed and incubated on ice for 20 min.500 μL of staining buffer was added to each sample, T cells werepelleted and resuspended in 200 μL buffer and analyzed on a flowcytometer (Becton Dickinson, FACScalibur) Intracellular staining wasconducted using the recommended procedure from the BD Pharmingen oreBioscience where indicated.

Example 10. Chromatin Immunoprecipitation-DNA Microarray (ChIP-Chip)

Human CD4+CD25hi Tregs were expanded in vitro as described previously(37).

Chromatin fixation and immunoprecipitation were performed usingchromatin immunoprecipitation assay kit (Upstate Biotechnology, Inc.) asrecommended by the manufacturer. Expanded human Treg were fixed in 1.1%formaldehyde. Protein-DNA cross-linked cell pellets were resuspended inSDS-Lysis Buffer (1 ml per IX 10⁸ cells) and incubated for IO minutes onice. Lysates were sonicated to shear DNA to lengths between 200 and I000 basepairs and centrifuged for 10 minutes at 13,000 rpm at 4° C. toremove debris. The sonicated cell supernatant was diluted IO-fold inChIP Dilution Buffer with protease inhibitors (Upstate Biotechnology,Inc) to reduce non-specific background, the diluted cell supernatant waspre-cleared with 40 μl of a Protein A Agarose-50% slurry per I ml lysatefor 30 minutes at 4° C. with agitation. Cross-linked protein/DNAcomplexes were immunoprecipitated using control rabbit lg oraffinity-purified rabbit polyclonal anti-human FoxP3 (a generous gift ofRoli Khattri and Fred Ramsdell, Celltech, Lt. Seattle, Wash.). Thecross-linking of the material was reversed and Proteinase K treated toremove protein from the DNA The remaining DNA was purified with QIAquickPCR Purification Kit (Qiagen, 28106) and amplified by LMPCR(ligation-mediated PCR) as described previously (49). Arrayhybridization and analysis were performed at Affymetrix as describedpreviously (50). SYBR Green qPCR was carried out to verify the bindingsites predicted by the arrays. Primers for the PCR reactions included:

IL-7R promoter: 5′-primer, (SEQ ID NO: 35) CAGGGAATATCCAGGAGGAA;3′-primer, (SEQ ID NO: 36) TGTGTGAGCCAGTGTGTATGAA; IL-7R 2K upstream:5′-primer, (SEQ ID NO: 37) TTTGGGATTTCTCCTTGAACA; 3′-primer(SEQ ID NO: 38) TCTCTGGGCATTTCAAAACC; IL-7R intron 4: 5′ primer,(SEQ ID NO: 39) GAGGTGGCAGAAGAGTGGAG; 3′-primer, (SEQ ID NO: 40)TGCATCACACTGCAAACAAA; IL7-R intron 7 and exon 8: 5′-primer,(SEQ ID NO: 41) ACATGCTGGCAATTCTGTGA; 3′-primer, (SEQ ID NO: 42)TCTGGCAGTCCAGGAAACTT.

Example 11. Lack of Correlation Between FoxP3 and CD25 in Human CD4+ TCells

Previous studies in mouse using FoxP3-GFP knock-in mice havedemonstrated that FoxP3 does not always correlate with CD25 expression(36). Since current efforts in humans have focused on the use of CD25 toisolate and quantify Tregs, we analyzed the expression of FoxP3 in thevarious CD4+ T cell subsets. Peripheral blood cells from normal subjectswere purified on Ficoll gradients and cell surface stained with anti-CD4and anti-CD25 mAbs. This staining was following by cell membranepermeabilization and intracellular staining with a monoclonal anti-FoxP3mAb.

As seen in FIG. 1 , although the majority of CD4+CD25hi cells (top 2% ofgate) were FoxP3+(ranging from 84.5-96.8% in 3 individuals) there wereconsiderable numbers of FoxP3+ cells that were CD25 dull or evennegative. In fact, based solely on CD4 and CD25 gating between 27-52.7%of the cells were FoxP3+ accounting for up to 7.5% of the CD4+ T cells.No significant staining was observed using an isotype control IgG-Alexa488 while a second anti-FoxP3 mAb from Biolegend gave similar results(data not shown).

An analysis of FoxP3 expression in the CD4+CD2S− T cell subset showedthat <5% of the CD25-CD4+ T cells expressed FoxP3, although thatpercentage was probably an overestimate due to some background stainingusing the isotype control lg. However, given the large numbers of cellsin this gate, there are likely to be at least some CD4+CD2S− T cellsthat are FoxP3+. Thus, rather than <2% of the CD4+ T cells falling intoa putative Treg subset, as many as 8-10% of the CD4+ T cells may beregulatory in nature.

Example 12. Analysis of Novel Treg-Specific Cell Surface Molecules

To identify additional cell surface markers associated with function andphenotype of Treg, microarray analysis was performed comparing mRNAexpressed by CD4+CD25hi T cells with CD4+cn25neg T cells isolated fromhealthy donor PBMC. mRNA was prepared from 3 blood donors, cRNA preparedand tested on Affymetrix U133A GeneChips. The sorting parameters werebased on published studies in which the top 1-2% of CD4+CD25+ T cellswere selected as the prototypic Treg subset (16, 37).

Among the genes that differed between the two subsets, IL-7R (CD127)expression was noted to be expressed at 2.4-fold lower levels inCD4+CD25hi T cells as compared to CD4+cn25neg T cells. To confirm thefindings, mRNA isolated from 3 independent CD4+CD25hi and CD4+cn25neg Tcell preparations were examined by quantitative real time PCR (qPCR).Expression of CD127 mRNA was inversely correlated with CD25 expression.In fact, the level of expression was 3.14 lower in the CD4+CD25hi Tcells as compared to CD4+cn25− T cells (range 2.26- to 4.21-fold).

As predicted by the gene expression studies, the majority of theCD4+CD25+ cells, especially the CD4+CD25hi T cells had low expression ofCD127 (FIG. 2 a ). However, not all of the CD4+CD12?1^(◯1)− T cells wereCD25+. In fact, a significant percentage of CD4+CD127− T cells (15.8% inthis individual) were CD25 negative. That said, the majority of theCD4+CD2S− T cells were CD127 bright (73.8%) accounting for thedifferential expression observed in the gene array analyses. Moreimportantly, flow cytometric analysis of FoxP3 expression in CD127positive and negative T cell subsets showed that the majority of FoxP3+T cells were in the CD12?1^(◯1)−− T cell subset (FIG. 2 b ).Interestingly, the relative expression of CD127 was inversely correlatedwith FoxP3 with the highest FoxP3-expressing CD4+ T cells expressing thelowest levels of CD127. These results were uniformly observed in >20different individuals examined. In fact, similar results were observedin mice. CD4+ T cells isolated for FoxP3-GFP knock-in mice were stainedfor CD127 (36, 38). The vast majority of the mouse CD4+FoxP3+ T cellswere CD12?1^(◯1)− (FIG. 3 a ). Additional analyses of these mice showedthat CD4+CD25+FoxP3+ Tregs were CD12?1^(◯1)⋅, however, like in humans,CD127 was a better marker than CD25 since all the CD4+ T cells wereCD12?1^(◯1)− independent of CD25 expression (FIG. 3 b ).

Further studies were conducted to determine the relationship of CD4,CD127, FoxP3 and CD25 using multiparameter flow cytometry (FIG. 4 ). Theoverwhelming majority of CD4+CD25+CD127lo/− T cells express FoxP3 (94%this individual) (FIG. 4 a ). However, a significant percentage ofCD4+CD25−CD12710/− cells are also FoxP3+(35% in this individual)although the mean fluorescence is often less than the CD4+CD25hi cells.In sharp contrast, there were few FoxP3+ T cells in the CD4+CD127+subset except a small percentage among those that expressed CD25. Thus,in spite of the fact that CD127+ T cells accounted for about 90% of theCD4+ T cells in this individual (FIG. 4 b ). Interestingly, back-gatingof the CD4+CD127+FoxP3+ subset showed that the expression of CD25 inthese T cells was intermediate and distinct from the CD25hi subsetdescribed as “classic” Tregs (data not shown) suggesting they may be atransitional cell. Similar results were observed in PBMCs obtained from10 healthy donors stained for cell surface expression of CD4, CD127,CD25 followed by intracellular staining with FoxP3-specific mAb (FIG. 4c ). Examination of multiple individuals confirmed that the majority ofthe CD4+FoxP3+ T cells were within the CD25+CD127lo/− subset, however,in some individuals a significant percentage of the CD25−CD127lo/−and/or CD25+CD127+ T cells were FoxP3+. Thus, CD127 is a better cellsurface marker than CD25 for the identification of CD4+FoxP3+ T cells,however, the best combination of cell surface markers isCD4+CD25+CD127lo/− which accounts for approximately 80% of the FoxP3+depending on the individual. Thus, a broad gating strategy ofCD4+CD25+CD127lo/− results in a highly purified FoxP3+ T cell populationas compared to the other subsets.

Example 13. ChIP-Chip Analysis of FoxP3 Interaction with CD127

The data clearly showed a “general” relationship between FoxP3expression and CD127 down-regulation. However, we were struck with theapparently inverse correlation between FoxP3 and CD127 protein (FIG. 2 b). These results suggested that there may be a direct structuralrelationship between the transcription factor, FoxP3, and CD127transcription. This was especially attractive given previous studiessuggesting that FoxP3 represses gene expression (39). Chromatin (Ch)immunoprecipitation (IP) of transcription factor-bound genomic DNAfollowed by microarray hybridization (chip) of IP-enriched DNA is a newtechnology that allows genome-wide analysis of transcription factorbinding. ChIP-chip data are different than classical microarray geneexpression data obtained by measuring mRNA levels in that it examinesdirect control of gene transcription not just potentially indirectdownstream regulation.

We performed ChIP-chip experiments on anti-CD3/anti-CD28-expandedCD4+CD25hi human Tregs (37). Anti-FoxP3 or control rabbit lg was used toprecipitate cross-linked protein-DNA complexes from nuclear lysates. Thecross-linking of the immunoprecipitated material was removed,protease-treated, and the DNA was purified and amplified. The resultantmaterial was hybridized to the whole genome using GeneChip® Human TilingI.OR Array Set (Affymetrix 900774) to identify the locations of bindingsites for FoxP3. Statistical analysis was performed to determine sitesthat were selectively associated with FoxP3 protein and not rabbit lg.

The IL-7R promoter region scored among the various sites bound by theFoxP3 protein immunoprecipitates (data not shown). The CD127 promoterbinding was confirmed by qPCR. Oligonucleotide primers spanning theCD127 promoter region were used on anti-FoxP3 immunoprecipitated DNAfrom CD4+CD25hi human Tregs (FIG. 5 a-5 b ). There was a strongenrichment of CD127 DNA amplified from the anti-FoxP3 immunoprecipitatesas compared to the rabbit lg immunoprecipitates specifically in theIL-7R promoter region but not other DNA sequences surrounding this areaon the genome. These data support the direct regulation of CD127 byFoxP3.

Example 14. Suppression of Allogeneic Mixed Lymphocyte Response (MLR)Using Different CD4+ T Cell Subsets

Although the low expression of CD127 correlated with FoxP3 expression, anumber of studies have questioned whether FoxP3 is always a marker ofTregs in humans. Thus, we examined the ability of CD4+CD25+CD12?1^(◯1)−T cells and other subsets to suppress an allogeneic MLR. PBMCs weresorted into 4 subsets based on CD127, CD25 and CD4 expression. First, weexamined the ability of the individual subsets to respond to theallogeneic APCs. As can be seen in FIG. 6 , as previously reported, theCD4+CD25hi T cell subset was anergic when stimulated with alloantigenconsistent with the fact that these cells were FoxP3+ and comprise the“classical” Treg subset. Similarly, neither of the CDI2?1^(◯1)− subsets,CD25+ or CD2S− CD4+ T cells or the bulk CD4+CDI2?1^(◯1)− T cellsresponded in the allogeneic MLR suggesting that like the CD4+CD25hi Tcells, these cells were anergic (37). In contrast, the CD4+CD127+ T cellsubset responded normally to alloantigen consistent with publicationssuggesting that these cells represented naive and memory T cellcompartments (32, 34, 40). Similar results were observed when the cellswere stimulated with anti-CD3 and anti-CD28 (data not shown) suggestingthat like “classical” Treg subsets, the FoxP3-expressing cells wereanergic. Next, the various subpopulations were added to an allogeneicMLR and compared for their ability to suppress T cell proliferation. TheCD4+CD12?1^(◯1)−cD2s+ T cell subset suppressed the MLR as well or betterthat CD4+CD25hi T cells (FIG. 7 a-7 b ). This is significant since thissubset represents at least 3-fold more CD4+ T cells including both CD25intermediate and negative subsets. Thus, CD127 is more that just anothermarker of CD4+cn25m Tregs but allows for the identification andisolation of a significantly more inclusive suppressive T cell subset.In fact, suppressive activity was independent of CD25 as both theCD4+CDI2?1^(◯1)−cn25+ and CD4+CDI2?1−cn2s− T cell subsets suppressed theMLR, although in multiple studies the CD4+cn12?1^(◯1)−cn25+ T cellssuppressed responses more effectively than the CD4+CD12?1^(◯1)−cn25− Tcell subset especially at lower Treg:Tresp ratios. These results areconsistent with the lower percentage and level of expression of FoxP3+cells in this T cell subset. Neither of the CD127+ cells suppressed theMLR reproducibly (n=9). As can be seen, the CD127 marker is the mostdescriminating in showing suppressor cell activity. These resultsindicated that CD127 is a sufficient marker for defining the CD4+regulatory T cell subset. To demonstrate this directly, PBMCs weresorted based only on the expression of CD4 and CD127 and examined in anallogeneic MLR. The CD4+cn12?1^(◯1)− T cell subset were anergic (datanot shown) and suppressed the MLR almost as efficiently as theCD4+CD127¹⁰¹⋅CD25+ or CD4+CD25hi T cells.

In addition, the Applicants compared gene expression profiles for a genearray in which mRNA from various T cell subsets were analyzed on an Affywhole genome chip and assessed for level of expression.CD4+CD127^(lo)CD25+ cells have closely the same overall fingerprint asthe classical CD4+CD25bright cells which differs from CD2S− cells (datanot shown), including, particularly with respect to selected T-regmarker TNF-R75, CTLA4, IL2RB, CD58, and CCR6 gene expression (notshown).

Example 15. Frequency of CD4+CD25+CD127¹⁰¹⋅T Cells from Patients withT1D

Previous studies have suggested that Treg numbers might be deficient inpatients with Type I diabetes (24). To investigate quantitativedifferences in the Treg populations in patients with TID versus controlsubjects, peripheral blood T cells were stained with CD4, CD25, CD127and FoxP3. The frequency of CD4+cn25+cn12?1^(◯1)− Tregs was notsignificantly different between TID (mean 66.1%, Std 11.3%, range53.7-82.8%) and control subjects (mean 65.5%, Std 8.66%, range45.4-76.2%) (FIG. 8 ). Although the percentages of FoxP3+ T cells amongthe CD4+ T cells in the two groups were higher than those reported forthe CD4+CD25hi Treg subset, the patterns matched those previouslyobserved. These results are in contrast with some reports that founddifferences in the percentage of CD4+CD25hi T cells in TID subjects whenanalyzed as a percentage of CD4+ T cells.

Example 16. CD4+cn12?1^(◯1)− Tregs are Suppressive and have TregPhenotype

Flow cytometric analyses showed that only 30-40% of the isolatedCD4+CD127lo cells expressed Foxp3 at the beginning of culture, with evenless Foxp3 expression (both based on percentage and MFI (meanfluorescence intensity)) after expansion. These results raised thequestion as to the functional activity of the Foxp3 negative T cells inthe culture. As a first approach to determining the functional potentialof the different cells grown in the culture, we separated CD4+CD127locells and cultured for 14 days with anti-CD3/anti-CD28+IL-2 plus/minusrapamycin. The cells expanded best in the absence of rapamycin and whenre-stimulated with the mAb and IL-2 cocktail on day 9. The cell expandedwith RAPA had the highest levels of Foxp3 and percentage of Foxp3+cells. The majority of Foxp3− cells were CD25lo versus Foxp3+ cells inthe same culture.

We next compared CD4+CD12?1^(◯) suppression for cells expanded in thepresence of IL-2 or IL-2 rapamycin. At 14 days, expanded cells wereseparated and added to a CFSE (carboxyfluorescein succinimidyl ester)suppression assay. Interestingly, after culture both populationssuppressed but those grown in Rapamycin suppressed better (see FIG. 12). This correlated with increased FoxP3 expression suggesting that FoxP3expression was linked to suppressive activity but not essential forsuppressor cell activity by the expanded cells. In this regard, itshould be noted in FIG. 7 that the fresh CD4+CD12?1^(◯)CD2S− T cellswere suppressive supporting the possibility that CD127, not Foxp3, is abetter marker for suppressor cell activity in this assay.

We next compared CD4+CD12?1^(◯) suppression based upon CD25 separationafter expansion (see, FIG. 13 a -FIG. 13 b ). At 14 days, expanded cellswere separated into CD25+ (middle column) and CD2S− (right column)subsets and added to a CFSE suppression assay. Interestingly, afterculture both populations suppressed equivalently suggesting that CD25expression was not essential to confer suppressive activity on theexpanded cells. However, we were unable to rule out whether the cellsthat suppressed in these cultures were derived from CD25+ cells or haddown-regulated CD25 during the culture.

We also examined FoxP3 and CD127 expression and Treg function of freshCD4+CD12?1^(◯) FoxP3+ and FoxP3− mouse T cells (see, FIG. 14 a -FIG. 14b ). These studies in mouse Tregs have confirmed our observations andshowed that only the FoxP3+CD127− cells suppress before expansion whileafter culture both both the FoxP3+ and FoxP3⋅ populations suppresseffectively. This result indicates that CD127 is the best marker andthat CD12?1^(◯) FoxP3+ cells can “educate” FoxP3− cells to becomesuppressors in vitro.

Discussion of the Examples

The above findings raise several critical issues. First, since themajority of CD4+FoxP3+ T cells may fall outside the typical gate forhuman Tregs, studies used for functional and immunophenotypic analysesare potentially missing a large number of putative Tregs. This hasimportant implications in determining quantitative differences inpatients with a variety of diseases. Second, the fact thatCD4+CD12?1^(◯1)−cD2S− T cells suppress an allogeneic MLR call intoquestion those studies suggesting that FoxP3 is not a “good” marker forTreg activity. It may be that FoxP3 is an excellent marker and the smallpopulations that arise during normal T cell activation are indeedadaptive regulatory T cells expanding as a consequence of sub optimal orsupra-optimal TCR signaling. It should be emphasized, however, that notall FoxP3+ T cells are necessarily Tregs and their activity may dependon the level of FoxP3 expression and isoforms of the protein expressed.However, these CD4+CD2s+cD12?1^(◯1)−, once isolated, may be treated invivo with TGF or other factors to enhance Treg function in these cells.Third, efforts to select Tregs for in vitro expansion may be hindered bythe underestimate of Tregs in any separation strategy based on CD25expression. The ability to identify and select a significantly greaternumber of Tregs circulating in the peripheral blood of humans,especially those with autoimmune diseases, is likely to make it easierto expand sufficient cell numbers for immunotherapy. Finally, theidentification of CD127 as a marker that distinguishes effector/memoryfrom regulatory T cells indicates that anti-CD127 therapy might beappropriate for the treatment of autoimmune diseases such as Type 1Diabetes, Systemic Lupus Erythematosis, or Multiple Sclerosis.

The identification of CD127 as a useful marker can be related to geneticobservations. First, microarray analysis of mRNA from individual T cellsubsets showed that CD127 was expressed at significantly lower levels inCD4+CD25hiversus CD4+CD2s− T cells. Unlike the majority of activated Tcells, which rapidly re-express CD127 and memory T cells that expresshigh levels of CD127, the Treg population remains CD12?1^(◯1)−. Theremay be two reasons for this. First, Tregs may be constantly undergoingantigenic stimulation which is CD28-dependent resulting in continuedsignaling that shuts down CD127 mRNA transcription. In this regard, itis interesting to note that activation of nai:ve T cells by anti-CD3plus anti-CD28 but not anti-CD3 alone led to a rapid down-regulation ofCD127 (J. Esensten, A. Weiss and J. A. Bluestone, unpublished data)suggesting that CD28 signals are uniquely involved in regulating CD127down-regulation. Alternatively, and not mutually exclusive, is thepossibility that FoxP3 expression in this T cell subset controls CD127expression. There are a number of reasons to think that this may be thecase As illustrated in the flow cytometric staining profiles, the moreFoxP3 expression, the less CD127 (FIG. 2 b ). In addition,over-expression of FoxP3 in transgenic mice results in a uniformlyCD12?1^(◯1)− population of cells with suppressive activity. Finally,data generated using CHIP analysis (first by ChIP-Chip followed byChIP-qPCR) suggested that the CD127 promoter is a target for FoxP3binding. Whether the low expression of CD127 is a consequence ofconstant antigen exposure in vivo or FoxP3 upregulation resulting inCD127 gene repression are not mutually exclusive.

The CD4+CD12?1^(◯1)−cD2S− T cells suppress quite effectively althoughthe percentage of FoxP3+ T cells in this subset can be quite variable.These results suggested that the CD127 marker may be useful inidentifying different subtypes of regulatory T cells including Tr1 andTH3 cells. In this regard, there are currently a number of settings,including the treatment of humans with T1D with anti-CD3 that induces Tcells with a regulatory phenotype (11, 31, 42, 43). These studies, whichmimic similar results in Treg-deficient mice treated with non-mitogenicanti-CD3 (10), indicate that it may be possible to identify an“adaptive” Treg response using CD127 as a biomarker in addition to lowerCD25 expression previously observed on these cells.

One of the more intriguing aspects of the results is the seemingdichotomy in cytokine receptor expression in memory T cells versusTregs. Whereas a high percentage of Tregs now appear to be IL-7R low andIL-2R positive, memory T cells have the opposite phenotype, expressinghigh levels of IL-7R and low levels of IL-2R. The functional basis forthis differential expression is unclear but it may reflect the evolutionof distinct pathways for cell survival and expansion of these T cellsubsets. For instance, it is possible that regulatory T cells may play acritical role in normal homeostasis. Thus, the cells attempt to regulatethe earliest immune perturbation that may occur in the absence of apathogenic response. Since IL-2 is an “early cytokine” produced rapidlyby activate T cells in the draining lymph nodes, IL-2 may be a criticalsignal for awakening the Treg response which can effectively suppress Tcell expansion in these lymphoid tissues (44). In contrast, IL-7 iscommonly produced locally in sites of inflammation leading to increasedsurvival and expansion of effector cells. If this localized IL-7expression promoted Treg expansion it might be counterproductive.Moreover, avoiding competition for the use of the common y chain bythese receptors would enhance the functionality of the cytokinefunction. Finally, it should be noted that the situation might be quitedistinct in thymus when all the pre-T and immature T cells are CD127+and CD25+. At this stage in development other factors might come intoplay to determine the differentiation pathways that determine whether aT cell become a Treg or a naive conventional T cell.

Several studies have examined the number and function of Treg cells inhumans with autoimmune diseases. In some settings, such as MultipleSclerosis, Type I Diabetes and autoimmune polyglandular syndrome II, thedata, based on the number and function of CD4+CD25hi Tregs, suggest thatthere are either fewer Tregs or less functional Tregs in diseasedindividuals (24, 45-47). However, in Type I Diabetes and otherautoimmune diseases, there have been contradictory results (25, 48). Inthe present study, we re-evaluated Tregs in patients with TID ascompared to normal individuals. Using the new markers, FoxP3 and CD127,we analyzed the frequency of CD4+CD25+FoxP3+CDI2?1^(◯1)⋅T cells and thefunction of sorted CD4+CD25+CDI2?1^(◯1)− to alloantigen stimulation. Inthis study, it is clear that human Tregs as defined by CD4, CD25, CD127and FoxP3 expression are present within the same range of percentages ascontrol individuals with no autoimmunity. Moreover, the functionality ofthe Tregs isolated from the patients with TID cannot be distinguishedfrom healthy control subjects. We cannot explain the basis fordifferences between our studies and those of others in the TID field. Ithad been suggested that the discrepancy might be due to subtledifferences in flow cytometry-based techniques for cell separation ordifferent mAbs used. However, our use of distinct markers that identifythe overwhelming bulk of Tregs in human peripheral blood is likely to bea more definitive assessment of the Treg numbers and functionalpotential in this patient population. Lastly, it is interesting to notethat several of the TID patients had high Tregs numbers as compared tothe bulk of the control and TID subjects. This is consistent with somestudies in other autoimmune diseases where the frequency of CD4+CD25hi Tcells was reported to be increased as compared to controls. Moreover,these results fit with mouse studies demonstrating increased Treg numberat the time of T1D disease onset as well as other immune diseasesettings (J. Adams, Q. Tang and J. A. Bluestone, unpublished data). Wehypothesize that rather than a Treg-deficiency being the cause ofdisease precipitation, there is actually increased Treg activity in anattempt to stem the losing battle being waged against the increasinglyaggressive effector cells that may indeed become more Treg resistant.

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes. No reference to a publication herein shouldbe construed as an admission that such is prior art.

What is claimed is:
 1. A method of making an isolated population ofimmunosuppressive regulatory T-cells, the method comprising: screening asample comprising human T cells for the levels of cell surfaceexpression of a CD4 marker and a CD127 marker to detect CD4⁺CD127^(lo/−)cells, wherein the CD4⁺CD127^(lo/−) cells have reduced or no levels ofcell surface-expressed CD127 compared to other cells in the sampleexpressing greater amounts of CD127; and isolating the CD4⁺CD127^(lo/−)cells from the sample to provide the isolated population ofimmunosuppressive regulatory T-cells.